3920_FM_i-xv 23/01/14 11:22 AM Page i Urinalysis and Body Fluids SIXTH EDITION Susan King Strasinger, DA, MLS(ASCP) Faculty Associate Clinical Laboratory Sciences Program The University of West Florida Pensacola, Florida Marjorie Schaub Di Lorenzo, BS, MLS(ASCP)SH Adjunct Instructor Division of Laboratory Sciences Clinical Laboratory Science Program University of Nebraska Medical Center Omaha, Nebraska and Phlebotomy Technician Program Coordinator Health Professions Nebraska Methodist College Omaha, Nebraska 3920_FM_i-xv 23/01/14 11:22 AM Page ii F. A. Davis Company 1915 Arch Street Philadelphia, PA 19103 www.fadavis.com Copyright © 2014 by F. A. Davis Company Copyright © 2014 by F. A. Davis Company. All rights reserved. This product is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America Last digit indicates print number: 10 9 8 7 6 5 4 3 2 1 Senior Acquisitions Editor: Christa Fratantoro Manager of Content Development: George W. Lang Developmental Editor: Molly Mullen Ward Art and Design Manager: Carolyn O’Brien As new scientific information becomes available through basic and clinical research, recommended treatments and drug therapies undergo changes. The author(s) and publisher have done everything possible to make this book accurate, up to date, and in accord with accepted standards at the time of publication. The author(s), editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of the book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised always to check product information (package inserts) for changes and new information regarding dose and contraindications before administering any drug. Caution is especially urged when using new or infrequently ordered drugs. Library of Congress Cataloging-in-Publication Data Strasinger, Susan King, author. Urinalysis and body fluids / Susan King Strasinger, Marjorie Schaub Di Lorenzo. — Sixth edition. p. ; cm. Includes bibliographical references and index. ISBN 978-0-8036-3920-1 (pbk. : alk. paper) I. Di Lorenzo, Marjorie Schaub, 1953- author. II. Title. [DNLM: 1. Urinalysis—methods. 2. Body Fluids—chemistry. QY 185] RB53 616.07’566—dc23 2013021830 Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by F. A. Davis Company for users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that the fee of $.25 per copy is paid directly to CCC, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payment has been arranged. The fee code for users of the Transactional Reporting Service is: 978-0-8036-3920-1/14 0 + $.25. 3920_FM_i-xv 23/01/14 11:22 AM Page iii To Harry—you will always be my Editor-in-Chief —SKS To my husband, Scott; my daughter, Lauren; my sons, Michael and Christopher; my daughters-in-law, Kathleen and Ashley; and my grandsons, Cameron and Joseph. —MSD 3920_FM_i-xv 23/01/14 11:22 AM Page iv 3920_FM_i-xv 23/01/14 11:22 AM Page v Preface As will be apparent to readers, the sixth edition of Urinalysis and Body Fluids has been substantially revised and enhanced. However, the objective of the text—to provide concise, comprehensive, and carefully structured instruction in the analysis of nonblood body fluids—remains the same. This sixth edition has been redesigned to meet the changes occurring in both laboratory medicine and instructional methodology. To meet the expanding technical information required by students in laboratory medicine, all of the chapters have been updated. Chapter 1 covers overall laboratory safety, precautions relating to urine and body fluid analysis, and the importance of quality assessment and management in the urinalysis laboratory. Preexamination, examination, and postexamination variables, procedure manuals, and current regulatory issues are stressed. Chapter 6 includes numerous additional images showing the various urine microscopic components. In Chapters 7 and 8 the most frequently encountered diseases of glomerular, tubular, interstitial, vascular, and hereditary origin are related to their associated laboratory tests. To accommodate advances in laboratory testing of cerebrospinal, seminal, synovial, serous, and amniotic fluids, all of the individual chapters have been enhanced, and additional anatomy and physiology sections have been added for each of these fluids. An entirely new chapter (Chapter 15) dedicated to vaginal secretions and covering proper specimen collection and handling, diseases, and related diagnosis laboratory tests has been added. Appendix A provides coverage of the ever-increasing variety of automated instrumentation available to the urinalysis laboratory. Appendix B discusses the analysis of bronchoalveolar lavage specimens, an area of the clinical laboratory that has been expanding in recent years. Each chapter opens with objectives and key terms and concludes with multiple choice questions for student review. In response to readers’ suggestions, the number of color images and figures has been significantly increased. The text has been extensively supplemented with tables, summaries, and procedure boxes. Case studies in the traditional format and clinical situations relating to technical considerations included at the end of the chapters offer students an opportunity to think critically about the material. A new feature, Historical Notes, provides a reference for topics or tests that are no longer routinely performed. Another new feature, Technical Tips, emphasizes information important to performing procedures. An answer key for the study questions, case studies, and clinical situations is included at the end of the book. Key terms appear in boldface blue color within the chapters. General medical terms appear in boldface in the text and are also included in the Glossary. The abbreviations noted in boldface red color have been collected in a convenient Abbreviations list at the back of the book. An electronic test bank, chapter-by-chapter PowerPoints, a searchable digital version of the textbook, resources for instructors, and interactive exercises and animations for students are provided on the DavisPlus Web site. We thank our readers for their valuable suggestions, which have guided us in creating this exciting new edition and the electronic ancillary supports. Susan King Strasinger Marjorie Schaub Di Lorenzo v 3920_FM_i-xv 23/01/14 11:22 AM Page vi 3920_FM_i-xv 23/01/14 11:22 AM Page vii Reviewers Lorraine Doucette, MS, MLS(ASCP)CM Associate Professor and Medical Laboratory Technician Program Coordinator Anne Arundel Community College Arnold, Maryland Pamela B. Lonergan, MS, MT(ASCP)SC Medical Technology Program Director Department of Nursing and Allied Health Norfolk State University Norfolk, Virginia Jessica Loontjer, MLS(ASCP)CM, LS(ASCP)CM Clinical Instructor, Special Chemistry and Urinalysis/Body Fluids Nebraska Methodist Hospital Laboratory Omaha, Nebraska Michelle Moy, MAd Ed, MT(ASCP)SC Program Director Clinical Laboratory Science Program Loyola University Chicago, Illinois C. Thomas Somma, PhD Associate Professor School of Medical Diagnostics and Translational Sciences College of Health Sciences Old Dominion University Norfolk, Virginia vii 3920_FM_i-xv 23/01/14 11:22 AM Page viii 3920_FM_i-xv 23/01/14 11:22 AM Page ix Acknowledgments Many people deserve credit for the help and encouragement they have provided us in the preparation of this sixth edition. Our continued appreciation is also extended to all of the people who were instrumental in the preparation of previous editions. The valuable suggestions from previous readers and the support from our colleagues at the University of West Florida, Northern Virginia Community College, University of Nebraska Medical Center, and Methodist Hospital have been a great asset to us in the production of this new edition. We thank each and every one of you. Brenda Franks has provided us with many valuable documents for reference in this text. The authors thank and acknowledge Pamela S. Hilke, MS, CT(ASCP), Education Coordinator and Instructor, and Sophie K. Thompson, MHS, CT, (ASCP) (IAC), Program Director and Associate Professor of the Cytotechnology Program at the School of Medical Diagnostic and Translational Sciences, College of Health Sciences, Old Dominion University, Norfolk, Virginia, for their contributions of spectacular cytology images. We extend special thanks to the people who have provided us with so many beautiful photographs for the text over the years: Donna L. Canterbury, BA, MT(ASCP)SH; Joanne M. Davis, BS, MT(ASCP)SH; M. Paula Neumann, MD; Gregory J. Swedo, MD; and Scott Di Lorenzo, DDS. We also thank Sherman Bonomelli, MS, for contributing original visual concepts that became the foundation for many of the line illustrations, and the students from the University of West Florida, specifically Jennifer Cardenas, Shannel Hill, Jelma Moore, and William Laguer, who worked under the guidance of Sherman Bonomelli to produce many of the new images. Images for Chapter 14 were provided by Carol Brennan, MT(ASCP), Diane Siedlik, MT(ASCP), Christian Herdt, MT(ASCP), and Teresa Karre, MD, from Methodist Hospital. We would like to express our gratitude for the help, patience, guidance, and understanding of our editors at F. A. Davis: Christa Fratantoro, Senior Acquisitions Editor; George Lang, Manager of Content Development, Health Professions/Medicine; and Molly M. Ward, Development Editor. We thank all the members of the F. A. Davis team who were instrumental in bringing this edition to fruition: Elizabeth Stepchin, Alisa Hathaway, Carolyn O’Brien, and Sharon Lee. ix 3920_FM_i-xv 23/01/14 11:22 AM Page x 3920_FM_i-xv 23/01/14 11:22 AM Page xi Contents PART ONE: Background CHAPTER 1 Safety and Quality Assessment 3 SAFETY 4 Biologic Hazards 4 Personal Protective Equipment 7 Hand Hygiene 7 Biologic Waste Disposal 9 Sharp Hazards 9 Chemical Hazards 10 Chemical Spills and Exposure 10 Chemical Handling 10 Chemical Hygiene Plan 10 Chemical Labeling 10 Material Safety Data Sheets 10 Radioactive Hazards 11 Electrical Hazards 11 Fire/Explosive Hazards 12 Physical Hazards 13 QUALITY ASSESSMENT 13 Urinalysis Procedure Manual 14 Preexamination Variables 14 Examination Variables 16 Postexamination Variables 20 CHAPTER 2 Introduction to Urinalysis 27 History and Importance 28 Urine Formation 29 Urine Composition 29 Urine Volume 29 Specimen Collection 30 Containers 30 Labels 30 Requisitions 31 Specimen Rejection 31 Specimen Handling 31 Specimen Integrity 31 Specimen Preservation 31 Types of Specimens 32 Random Specimen 32 First Morning Specimen 33 24-Hour (or Timed) Specimen 33 Catheterized Specimen 34 Midstream Clean-Catch Specimen 34 Suprapubic Aspiration 34 Prostatitis Specimen 34 Pediatric Specimens 34 Drug Specimen Collection 35 CHAPTER 3 Renal Function 39 Renal Physiology 40 Renal Blood Flow 40 Glomerular Filtration 41 Tubular Reabsorption 43 Tubular Secretion 45 Renal Function Tests 46 Glomerular Filtration Tests 47 Cystatin C 49 Tubular Reabsorption Tests 50 Tubular Secretion and Renal Blood Flow Tests 52 PART TWO: Urinalysis CHAPTER 4 Physical Examination of Urine 59 Color 60 Normal Urine Color 60 Abnormal Urine Color 61 Clarity 62 Normal Clarity 62 Nonpathologic Turbidity 63 Pathologic Turbidity 63 Specific Gravity 63 Refractometer 64 Osmolality 65 Reagent Strip Specific Gravity 66 Odor 66 CHAPTER 5 Chemical Examination of Urine 71 Reagent Strips 72 Reagent Strip Technique 72 Handling and Storing Reagent Strips 73 Quality Control of Reagent Strips 73 Confirmatory Testing 73 pH 73 Clinical Significance 73 Reagent Strip Reactions 75 Protein 75 Clinical Significance 75 Prerenal Proteinuria 75 xi 3920_FM_i-xv 23/01/14 11:22 AM Page xii xii Contents Renal Proteinuria 76 Postrenal Proteinuria 76 Reagent Strip Reactions 77 Reaction Interference 77 Glucose 79 Clinical Significance 79 Reagent Strip (Glucose Oxidase) Reaction 81 Reaction Interference 81 Copper Reduction Test (Clinitest) 81 Clinical Significance of Clinitest 82 Ketones 82 Clinical Significance 82 Reagent Strip Reactions 83 Reaction Interference 83 Blood 83 Clinical Significance 84 Hematuria 84 Hemoglobinuria 84 Myoglobinuria 84 Reagent Strip Reactions 84 Reaction Interference 85 Bilirubin 85 Bilirubin Production 85 Clinical Significance 86 Reagent Strip (Diazo) Reactions 87 Reaction Interference 87 Urobilinogen 87 Clinical Significance 88 Reagent Strip Reactions and Interference 88 Reaction Interference 88 Nitrite 88 Clinical Significance 88 Reagent Strip Reactions 89 Reaction Interference 89 Leukocyte Esterase 90 Clinical Significance 90 Reagent Strip Reaction 90 Reaction Interference 91 Specific Gravity 91 Reagent Strip Reaction 91 Reaction Interference 92 CHAPTER 6 CHAPTER 7 Renal Disease 147 Glomerular Disorders 148 Glomerulonephritis 148 Nephrotic Syndrome 149 Tubular Disorders 150 Acute Tubular Necrosis 150 Hereditary and Metabolic Tubular Disorders Interstitial Disorders 154 Acute Pyelonephritis 155 Chronic Pyelonephritis 155 Acute Interstitial Nephritis 155 Renal Failure 155 Renal Lithiasis 157 Urine Screening for Metabolic Disorders 163 Overflow Versus Renal Disorders 164 Newborn Screening Tests 164 Amino Acid Disorders 165 Phenylalanine-Tyrosine Disorders 165 Branched-Chain Amino Acid Disorders 167 Tryptophan Disorders 168 Cystine Disorders 169 PART THREE: Other Body Fluids CHAPTER 9 Cerebrospinal Fluid 181 101 153 CHAPTER 8 Porphyrin Disorders 170 Mucopolysaccharide Disorders 172 Purine Disorders 174 Carbohydrate Disorders 174 Microscopic Examination of Urine 99 Macroscopic Screening 100 Specimen Preparation 100 Specimen Volume 100 Centrifugation 100 Sediment Preparation 101 Volume of Sediment Examined 101 Commercial Systems 101 Examining the Sediment 101 Reporting the Microscopic Examination Correlating Results 102 Sediment Examination Techniques 102 Sediment Stains 103 Cytodiagnostic Urine Testing 105 Microscopy 105 Types of Microscopy 107 Urine Sediment Constituents 110 Red Blood Cells 110 White Blood Cells 112 Epithelial Cells 113 Bacteria 118 Yeast 119 Parasites 119 Spermatozoa 120 Mucus 120 Casts 121 Urinary Crystals 128 Urinary Sediment Artifacts 138 Formation and Physiology 182 Specimen Collection and Handling 182 3920_FM_i-xv 23/01/14 11:22 AM Page xiii Contents Appearance 183 Traumatic Collection (Tap) 184 Uneven Blood Distribution 184 Clot Formation 184 Xanthochromic Supernatant 185 Cell Count 185 Methodology 185 Total Cell Count 186 WBC Count 186 Quality Control of CSF and Other Body Fluid Cell Counts 186 Differential Count on a CSF Specimen 186 Cytocentrifugation 186 CSF Cellular Constituents 187 Chemistry Tests 193 Cerebrospinal Protein 193 CSF Glucose 196 CSF Lactate 195 CSF Glutamine 195 Microbiology Tests 195 Gram Stain 196 Serologic Testing 197 CHAPTER 10 Semen 203 Physiology 204 Specimen Collection 205 Specimen Handling 205 Semen Analysis 205 Appearance 205 Liquefaction 206 Volume 206 Viscosity 206 pH 207 Sperm Concentration and Sperm Count 207 Sperm Motility 208 Sperm Morphology 209 Additional Testing 210 Sperm Vitality 211 Seminal Fluid Fructose 211 Antisperm Antibodies 212 Microbial and Chemical Testing 212 Postvasectomy Semen Analysis 213 Sperm Function Tests 213 Semen Analysis Quality Control 213 CHAPTER 11 Synovial Fluid 217 Physiology 218 Specimen Collection and Handling 218 Color and Clarity 219 Viscosity 219 Cell Counts 220 Differential Count 220 Crystal Identification 221 Types of Crystals 221 Slide Preparation 222 Crystal Polarization 222 Chemistry Tests 224 Microbiologic Tests 224 Serologic Tests 224 CHAPTER 12 Serous Fluid 229 Formation 230 Specimen Collection and Handling 230 Transudates and Exudates 231 General Laboratory Procedures 231 Pleural Fluid 232 Appearance 232 Hematology Tests 232 Chemistry Tests 235 Microbiologic and Serologic Tests 236 Pericardial Fluid 236 Appearance 237 Laboratory Tests 237 Peritoneal Fluid 237 Transudates Versus Exudates 237 Appearance 238 Laboratory Tests 238 CHAPTER 13 Amniotic Fluid 243 Physiology 244 Function 244 Volume 244 Chemical Composition 244 Differentiating Maternal Urine From Amniotic Fluid 245 Specimen Collection 245 Indications for Amniocentesis 245 Collection 246 Specimen Handling and Processing 246 Color and Appearance 246 Tests for Fetal Distress 246 Hemolytic Disease of the Newborn 246 Neural Tube Defects 247 Tests for Fetal Maturity 248 Fetal Lung Maturity 248 Lecithin-Sphingomyelin Ratio 248 Phosphatidyl Glycerol 249 Foam Stability Index 249 Lamellar Bodies 249 xiii 3920_FM_i-xv 23/01/14 11:22 AM Page xiv xiv Contents CHAPTER 14 Fecal Analysis 255 Physiology 256 Diarrhea and Steatorrhea 257 Diarrhea 257 Steatorrhea 258 Specimen Collection 258 Macroscopic Screening 258 Color 258 Appearance 259 Microscopic Examination of Feces 259 Fecal Leukocytes 259 Muscle Fibers 259 Qualitative Fecal Fats 260 Chemical Testing of Feces 261 Occult Blood 261 Quantitative Fecal Fat Testing 262 APT Test (Fetal Hemoglobin) 263 Fecal Enzymes 264 Carbohydrates 264 CHAPTER 15 Vaginal Secretions 269 Specimen Collection and Handling 270 Color and Appearance 271 Diagnostic Tests 271 pH 271 Microscopic Procedures 272 Vaginal Disorders 277 Bacterial Vaginosis 277 Trichomoniasis 278 Candidiasis 278 Desquamative Inflammatory Vaginitis 279 Atrophic Vaginitis 279 Additional Vaginal Secretion Procedures 279 Fetal Fibronectin Test 279 AmniSure Test 279 Urine and Body Fluid Analysis Automation 283 APPENDIX B Bronchoalveolar Lavage 293 Answers to Study Questions and Case Studies and Clinical Situations 297 Abbreviations 305 Glossary 307 APPENDIX A Index 315 3920_FM_i-xv 23/01/14 11:22 AM Page xv 3920_Ch01_002-026 23/01/14 9:19 AM Page 2 PART ONE Background Chapter 1: Safety and Quality Assessment Chapter 2: Introduction to Urinalysis Chapter 3: Renal Function 3920_Ch01_002-026 23/01/14 9:19 AM Page 3 CHAPTER 1 Safety and Quality Assessment LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 1-1 List the six components of the chain of infection and the laboratory safety precautions that break the chain. 1-7 Discuss the components and purpose of chemical hygiene plans and Material Safety Data Sheets. 1-2 State the purpose of the Standard Precautions policy and describe its guidelines. 1-8 State and interpret the components of the National Fire Protection Association hazardous material labeling system. 1-3 State the requirements mandated by the Occupational Exposure to Blood-Borne Pathogens Compliance Directive. 1-4 Describe the types of personal protective equipment that laboratory personnel wear, including when, how, and why each article is used. 1-5 Correctly perform hand hygiene procedures following Centers for Disease Control and Prevention (CDC) guidelines. 1-6 Describe the acceptable methods for handling and disposing of biologic waste and sharp objects in the urinalysis laboratory. 1-9 Describe precautions that laboratory personnel should take with regard to radioactive, electrical, and fire hazards. 1-10 Explain the RACE and PASS actions to be taken when a fire is discovered. 1-11 Recognize standard hazard warning symbols. 1-12 Define the preexamination, examination, and postexamination components of quality assessment. 1-13 Distinguish between the components of internal quality control, external quality control, electronic quality control, and proficiency testing. KEY TERMS Accreditation External quality assessment (EQA) Postexposure prophylaxis (PEP) Accuracy External quality control Precision Biohazardous Fomite Preexamination variable Chain of infection Infection control Preventive maintenance (PM) Chemical hygiene plan Internal quality control Proficiency testing Clinical Laboratory Improvement Amendments (CLIA) Material Safety Data Sheet (MSDS) Quality assessment (QA) Occupational Safety and Health Administration (OSHA) Quality control (QC) Electronic quality control Personal protective equipment (PPE) Reliability Examination variable Postexamination variable Clinical and Laboratory Standards Institute (CLSI) Radioisotope Standard Precautions Turnaround time (TAT) 3920_Ch01_002-026 23/01/14 9:19 AM Page 4 4 Part One | Background S A F E T Y The clinical laboratory contains a variety of safety hazards, many of which are capable of producing serious injury or lifethreatening disease. To work safely in this environment, laboratory personnel must learn what hazards exist, the basic safety precautions associated with them, and how to apply the basic rules of common sense required for everyday safety for patients, co-workers, and themselves. As can be seen in Table 1–1, some hazards are unique to the health-care environment, and others are encountered routinely throughout life. Safety procedure manuals must be readily available in the laboratory that describe the safety policies mandated by the Centers for Disease Control and Prevention (CDC) and the Occupational Safety and Health Administration (OSHA), and strict adherence to these guidelines by laboratory personnel is essential. The manual must be updated and reviewed annually by the laboratory director. The Clinical and Laboratory Standards Institute (CLSI) provides the guidelines for writing these procedures and policies.1-3 Biologic Hazards The health-care setting provides abundant sources of potentially harmful microorganisms. These microorganisms are frequently present in the specimens Table 1–1 Types of Safety Hazards Type Source Possible Injury Biologic Infectious agents Bacterial, fungal, viral, or parasitic infections Cuts, punctures, or blood-borne pathogen exposure Exposure to toxic, carcinogenic, or caustic agents Radiation exposure Sharps Needles, lancets, broken glass Chemical Preservatives and reagents Radioactive Equipment and radioisotopes Ungrounded or wet equipment; frayed cords Open flames, organic chemicals Wet floors, heavy boxes, patients Electrical Fire/ explosive Physical Burns or shock Burns or dismemberment Falls, sprains, or strains From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, third edition, FA Davis, Philadelphia, 2011, p 52, with permission. received in the clinical laboratory. Understanding how microorganisms are transmitted (chain of infection) is essential to preventing infection. All health-care facilities have developed procedures to control and monitor infections occurring within their facilities. This is referred to as infection control. The chain of infection requires a continuous link between an infectious agent, a reservoir, a portal of exit, a means of transmission, a portal of entry, and a susceptible host.4 Infectious agents consist of bacteria, fungi, parasites, and viruses. The reservoir is the location of potentially harmful microorganisms, such as a contaminated clinical specimen or an infected patient. It is the place where the infectious agent can live and possible multiply. Humans and animals make excellent reservoirs. Equipment and other soiled inanimate objects, called fomites, will serve as reservoirs, particularly if they contain blood, urine, or other body fluids. Some microorganisms form spores or become inactive when conditions are not ideal and wait until a suitable reservoir is available. The infectious agent must have a way to exit the reservoir to continue the chain of infection. This can be through the mucous membranes of the nose, mouth, and eyes, and in blood or other body fluids. Once the infectious agent has left the reservoir, it must have a way to reach a susceptible host. Means of transmission include: 1. Direct contact: the unprotected host touches the patient, specimen, or a contaminated object (reservoir) 2. Airborne: inhalation of dried aerosol particles circulating on air currents or attached to dust particles 3. Droplet: the host inhales material from the reservoir (e.g., aerosol droplets from a patient or an uncapped centrifuge tube, or when specimens are aliquoted or spilled) 4. Vehicle: ingestion of a contaminated substance (e.g., food, water, specimen) 5. Vector: from an animal or insect bite After the infectious agent has been transmitted to a new reservoir, it must have a means to enter the reservoir. The portal of entry can be the same as the portal of exit, which includes the mucous membranes of the nose, mouth, and eyes, breaks in the skin, and open wounds. The susceptible host can be another patient during invasive procedures, visitors, and healthcare personnel when exposed to infectious specimens or needlestick injuries. Immunocompromised patients, newborns and infants, and the elderly are often more susceptible hosts. Stress, fatigue, and lack of proper nutrition depress the immune system and contribute to the susceptibility of patients and health-care providers. Once the chain of infection is complete, the infected host then becomes another source able to transmit the microorganisms to others.1 In the clinical laboratory, the most direct contact with a source of infection is through contact with patient specimens, although contact with patients and infected objects also occurs. Preventing completion of the chain of infection is a primary objective of biologic safety. Figure 1–1 illustrates the universal symbol for biohazardous material and demonstrates how following prescribed safety practices can break the chain of infection. Figure 1–1 places particular emphasis on laboratory practices. 3920_Ch01_002-026 23/01/14 9:19 AM Page 5 Chapter 1 | Safety and Quality Assessment Break the link • Immunizations • Patient isolation • Nursery precautions • Healthy lifestyle Susceptible host • Patients • Elderly • Newborns • Immunocompromised • Health-care workers Break the link • Disinfection • Hand hygiene Infectious agent • Bacteria • Fungi • Parasites • Viruses Reservoir • Humans • Animals • Insects • Fomites • Blood/body fluids Portal of exit • Nose • Mouth • Mucous membranes • Specimen collection Portal of entry • Nose • Mouth • Mucous membranes • Skin • Unsterile equipment Break the link • Hand hygiene • Standard precautions • PPE • Sterile equipment 5 Means of transmission • Droplet • Airborne • Contact • Vector • Vehicle Break the link • Sealed biohazardous waste containers • Sealed specimen containers • Hand hygiene • Standard precautions Break the link • Hand hygiene • Standard precautions • PPE • Patient isolation Figure 1–1 Chain of infection and safety practices related to the biohazard symbol. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, FA Davis, Philadelphia, 2011, with permission.) Proper hand hygiene, correct disposal of contaminated materials, and wearing personal protective equipment (PPE) are of major importance in the laboratory. Concern over exposure to blood-borne pathogens, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV), resulted in the drafting of guidelines and regulations by the CDC and OSHA to prevent exposure. In 1987 the CDC instituted Universal Precautions (UP). Under UP all patients are considered to be possible carriers of bloodborne pathogens. The guideline recommends wearing gloves when collecting or handling blood and body fluids contaminated with blood and wearing face shields when there is danger of blood splashing on mucous membranes and when disposing of all needles and sharp objects in puncture-resistant containers. The CDC excluded urine and body fluids not visibly contaminated by blood from UP, although many specimens can contain a considerable amount of blood before it becomes visible. The modification of UP for body substance isolation (BSI) helped to alleviate this concern. BSI guidelines are not limited to blood-borne pathogens; they consider all body fluids and moist body substances to be potentially infectious. According to BSI guidelines, personnel should wear gloves at all times when encountering moist body substances. A major disadvantage of BSI guidelines is that they do not recommend handwashing after removing gloves unless visual contamination is present. 3920_Ch01_002-026 23/01/14 9:19 AM Page 6 6 Part One | Background In 1996 the CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC) combined the major features of UP and BSI guidelines and called the new guidelines Standard Precautions. Although Standard Precautions, as described below, stress patient contact, the principles can also be applied to handling patient specimens in the laboratory.5 Standard Precautions are as follows: 1. Hand hygiene: Hand hygiene includes both hand washing and the use of alcohol-based antiseptic cleansers. Sanitize hands after touching blood, body fluids, secretions, excretions, and contaminated items, whether or not gloves are worn. Sanitize hands immediately after gloves are removed, between patient contacts, and when otherwise indicated to avoid transferring microorganisms to other patients or environments. Sanitizing hands may be necessary between tasks and procedures on the same patient to prevent crosscontamination of different body sites. 2. Gloves: Wear gloves (clean, nonsterile gloves are adequate) when touching blood, body fluids, secretions, excretions, and contaminated items. Put on gloves just before touching mucous membranes and nonintact skin. Change gloves between tasks and procedures on the same patient after contact with material that may contain a high concentration of microorganisms. Remove gloves promptly after use, before touching noncontaminated items and environmental surfaces, and between patients. Always sanitize your hands immediately after glove removal to avoid transferring microorganisms to other patients or environments. 3. Mouth, nose, and eye protection: Wear a mask and eye protection or a face shield to protect mucous membranes of the eyes, nose, and mouth during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. A specially fitted respirator (N95) must be used during patient care activities related to suspected mycobacterium exposure. 4. Gown: Wear a gown (a clean, nonsterile gown is adequate) to protect skin and to prevent soiling of clothing during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. Select a gown that is appropriate for the activity and the amount of fluid likely to be encountered (e.g., fluid-resistant in the laboratory). Remove a soiled gown as promptly as possible, and sanitize hands to avoid transferring microorganisms to other patients or environments. 5. Patient care equipment: Handle used patient care equipment soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposure, clothing contamination, and transfer of microorganisms to other patients or environments. Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. Ensure that single-use items are discarded properly. 6. Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces, beds, bedrails, bedside equipment, and other frequently touched surfaces. Ensure that these procedures are being followed. 7. Linen: Handle, transport, and process linen soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures and clothing contamination and that avoids the transfer of microorganisms to other patients and environments. 8. Occupational health and blood-borne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices; when handling sharp instruments after procedures; when cleaning used instruments; and when disposing of used needles. Never recap used needles or otherwise manipulate them using both hands or use any other technique that involves directing the point of a needle toward any part of the body; rather, use self-sheathing needles or a mechanical device to conceal the needle. Do not remove used unsheathed needles from disposable syringes by hand, and do not bend, break, or otherwise manipulate used needles by hand. Place used disposable syringes and needles, scalpel blades, and other sharp items in appropriate puncture-resistant containers, which are located as close as practical to the area in which the items were used, and place reusable syringes and needles in a puncture-resistant container for transport to the reprocessing area. Use mouthpieces, resuscitation bags, or other ventilation devices as an alternative to mouthto-mouth resuscitation methods in areas where the need for resuscitation is predictable. 9. Patient placement: Place a patient in a private room who contaminates the environment or who does not (or cannot be expected to) assist in maintaining appropriate hygiene or environment control. If a private room is not available, consult with infection control professionals regarding patient placement or other alternatives. 10. Respiratory hygiene/cough etiquette: Educate health-care personnel, patients, and visitors to contain respiratory secretions to prevent droplet and fomite transmission of respiratory pathogens. Offer masks to coughing patients, distance symptomatic patients from others, and practice good hand hygiene to prevent the transmission of respiratory pathogens. The Occupational Exposure to Blood-Borne Pathogens Standard is a law monitored and enforced by OSHA.6,7 These controls are required by OSHA to be provided by or mandated by the employer for all employees. Specific requirements of this OSHA standard include the following: Engineering Controls 1. Providing sharps disposal containers and needles with safety devices. 3920_Ch01_002-026 23/01/14 9:19 AM Page 7 Chapter 1 | Safety and Quality Assessment 2. Requiring discarding of needles with the safety device activated and the holder attached. 3. Labeling all biohazardous materials and containers. Work Practice Controls 4. Requiring all employees to practice Standard Precautions and documenting training on an annual basis. 5. Prohibiting eating, drinking, smoking, and applying cosmetics in the work area. 6. Establishing a daily work surface disinfection protocol. Personal Protective Equipment 7. Providing laboratory coats, gowns, face shields, and gloves to employees and laundry facilities for nondisposable protective clothing. Medical 8. Providing immunization for the hepatitis B virus free of charge. 9. Providing medical follow-up to employees who have been accidentally exposed to blood-borne pathogens. Documentation 10. Documenting annual training of employees in safety standards. 11. Documenting evaluations and implementation of safer needle devices. 12. Involving employees in the selection and evaluation of new devices and maintaining a list of those employees and the evaluations. 13. Maintaining a sharps injury log including the type and brand of safety device, location and description of the incident, and confidential employee follow-up. Any accidental exposure to a possible blood-borne pathogen must be immediately reported to a supervisor. Evaluation of the incident must begin right away to ensure appropriate postexposure prophylaxis (PEP). The CDC provides periodically updated guidelines for the management of exposures and recommended PEP.8,9 Personal Protective Equipment PPE used in the laboratory includes gloves, fluid-resistant gowns, eye and face shields, and Plexiglas countertop shields. Gloves should be worn when in contact with patients, specimens, and laboratory equipment or fixtures. When specimens are collected, gloves must be changed between every patient. In the laboratory, they are changed whenever they become noticeably contaminated or damaged and are always removed when leaving the work area. Wearing gloves is not a substitute for hand hygiene, and hands must be sanitized after gloves are removed. A variety of gloves types are available, including sterile and nonsterile, powdered and unpowdered, and latex and nonlatex. Allergy to latex is increasing among health-care workers, and laboratory personnel should be alert for symptoms of reactions associated with latex. Reactions to latex include irritant contact 7 dermatitis, which produces patches of dry, itchy irritation on the hands; delayed hypersensitivity reactions resembling poison ivy that appear 24 to 48 hours after exposure; and true, immediate hypersensitivity reactions often characterized by facial flushing and breathing difficulties. Hand sanitizing immediately after removing gloves and avoiding powdered gloves may aid in preventing the development of latex allergies. Replacing latex gloves with nitrile or vinyl gloves provides an alternative. Any symptoms of latex allergy should be reported to a supervisor because true latex allergy can be life-threatening.10 Fluid-resistant laboratory coats with wrist cuffs are worn to protect clothing and skin from exposure to patients’ body substances. These coats should always be completely buttoned, and gloves should be pulled over the cuffs. They are worn at all times when working with patient specimens and are removed prior to leaving the work area. They are changed when they become visibly soiled. Disposable coats are placed in containers for biohazardous waste, and nondisposable coats are placed in designated laundry receptacles. Shoes must be closed-toed and cover the entire foot. The mucous membranes of the eyes, nose, and mouth must be protected from specimen splashes and aerosols. A variety of protective equipment is available, including masks and goggles, full-face plastic shields that cover the front and sides of the face, mask with attached shield, and Plexiglas countertop shields. Particular care should be taken to avoid splashes and aerosols when removing container tops, pouring specimens, and centrifuging specimens. Specimens must never be centrifuged in uncapped tubes or in uncovered centrifuges. When specimens are received in containers with contaminated exteriors, the exterior of the container must be disinfected or, if necessary, a new specimen may be requested. Hand Hygiene Hand hygiene is emphasized in Figure 1–1 and in the Standard Precautions guidelines. Hand contact is the primary method of infection transmission. Laboratory personnel must always sanitize hands before patient contact, after gloves are removed, before leaving the work area, at any time when hands have been knowingly contaminated, before going to designated break areas, and before and after using bathroom facilities. Hand hygiene includes both hand washing and using alcoholbased antiseptic cleansers. Alcohol-based cleansers can be used when hands are not visibly contaminated. They are not recommended after contact with spore-forming bacteria, including Clostridium difficile and Bacillus sp. When using alcohol-based cleansers, apply the cleanser to the palm of one hand. Rub your hands together and over the entire cleansing area, including between the fingers and thumbs. Continue rubbing until the alcohol dries. The CDC has developed hand washing guidelines to be followed for correct hand washing.1,11 Procedure 1-1 demonstrates CDC routine hand washing guidelines.4 More stringent procedures are used in surgery and in areas with highly susceptible patients, such as immunocompromised and burn patients. 3920_Ch01_002-026 23/01/14 9:20 AM Page 8 8 Part One | Background PROCEDURE 1-1 Hand Washing Procedure Equipment Antimicrobial soap Paper towels Running water Waste container Procedure 1. Wet hands with warm water. Do not allow parts of body to touch the sink. 3. Rub to form a lather, create friction, and loosen debris. Thoroughly clean between the fingers and under the fingernails for at least 20 seconds; include thumbs and wrists in the cleaning. 4. Rinse hands in a downward position to prevent recontamination of hands and wrists. 2. Apply soap, preferably antimicrobial. 5. Obtain paper towel from the dispenser. 3920_Ch01_002-026 23/01/14 9:20 AM Page 9 Chapter 1 | Safety and Quality Assessment 9 PROCEDURE 1-1—cont’d 6. Dry hands with paper towel. 7. Turn off faucets with a clean paper towel to prevent contamination. Biologic Waste Disposal All biologic waste, except urine, must be placed in appropriate containers labeled with the biohazard symbol (Fig. 1–2). This includes both specimens and the materials with which the specimens come in contact. The waste is then decontaminated following institutional policy: incineration, autoclaving, or pickup by a certified hazardous waste company. Urine may be discarded by pouring it into a laboratory sink under a Plexiglas countertop shield. Care must be taken to avoid splashing, and the sink should be flushed with water after specimens are discarded. Disinfection of the sink using a 1:5 or 1:10 dilution of sodium hypochlorite should be performed daily. Sodium hypochlorite dilutions stored in plastic bottles are effective for 1 month if protected from light after preparation.12 The same solution also can be used for routinely disinfecting countertops and accidental spills. The solution should be allowed to air-dry on the contaminated area. Absorbent materials used for cleaning countertops and removing spills must be discarded in biohazard containers. Empty urine containers can be discarded as nonbiologically hazardous waste (Fig. 1–3). Sharp Hazards Sharp objects in the laboratory, including needles, lancets, and broken glassware, present a serious biologic hazard, particularly for the transmission of blood-borne pathogens. All sharp objects must be disposed in puncture-resistant, leak-proof container with the biohazard symbol. Puncture-resistant containers should be conveniently located within the work area. The biohazard sharp containers should not be overfilled and must always be replaced when the safe capacity mark is reached. Figure 1–2 Biohazard symbol. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, FA Davis, Philadelphia, 2011, with permission.) 3920_Ch01_002-026 23/01/14 9:20 AM Page 10 10 Part One | Background removed as soon as possible. No attempt should be made to neutralize chemicals that come in contact with the skin. Chemical spill kits containing protective apparel, nonreactive absorbent material, and bags for disposing of contaminated materials should be available for cleaning up spills. Chemical Handling Chemicals should never be mixed together unless specific instructions are followed, and they must be added in the order specified. This is particularly important when combining acid and water. Acid should always be added to water to avoid the possibility of sudden splashing caused by the rapid generation of heat in some chemical reactions. Wearing goggles and preparing reagents under a fume hood are recommended safety precautions. Chemicals should be used from containers that are of an easily manageable size. Pipetting by mouth is unacceptable in the laboratory. State and federal regulations are in place for the disposal of chemicals and should be consulted. A Chemical Hygiene Plan OSHA also requires all facilities that use hazardous chemicals to have a written chemical hygiene plan (CHP) available to employees.13 The purpose of the plan is to detail the following: 1. Appropriate work practices 2. Standard operating procedures 3. PPE 4. Engineering controls, such as fume hoods and flammables safety cabinets 5. Employee training requirements 6. Medical consultation guidelines Each facility must appoint a chemical hygiene officer, who is responsible for implementing and documenting compliance with the plan. Examples of required safety equipment and information are shown in Figure 1–4. B Figure 1–3 Technologist disposing of urine (A) sample and (B) container. Chemical Hazards The same general rules for handling biohazardous materials apply to chemically hazardous materials; that is, to avoid getting these materials in or on bodies, clothes, or work area. Every chemical in the workplace should be presumed hazardous. Chemical Spills and Exposure When skin contact occurs, the best first aid is to flush the area with large amounts of water for at least 15 minutes, then seek medical attention. For this reason, all laboratory personnel should know the location and proper use of emergency showers and eye wash stations. Contaminated clothing should be Chemical Labeling Hazardous chemicals should be labeled with a description of their particular hazard, such as poisonous, corrosive, flammable, explosive, teratogenic, or carcinogenic (Fig. 1–5). The National Fire Protection Association (NFPA) has developed the Standard System for the Identification of the Fire Hazards of Materials, NFPA 704.14 This symbol system is used to inform firefighters of the hazards they may encounter with fires in a particular area. The diamond-shaped, color-coded symbol contains information relating to health, flammability, reactivity, and personal protection/special precautions. Each category is graded on a scale of 0 to 4, based on the extent of concern. These symbols are placed on doors, cabinets, and containers. An example of this system is shown in Figure 1–6. Material Safety Data Sheets The OSHA Federal Hazard Communication Standard requires that all employees have a right to know about all chemical hazards present in their workplace. The information is provided 3920_Ch01_002-026 23/01/14 9:20 AM Page 11 Chapter 1 | Safety and Quality Assessment 11 in the form of Material Safety Data Sheets (MSDSs) on file in the workplace. By law, vendors are required to provide these sheets to purchasers; however, the facility itself is responsible for obtaining and making MSDSs available to employees. Information contained in an MSDS includes the following: 1. Physical and chemical characteristics 2. Fire and explosion potential 3. Reactivity potential 4. Health hazards and emergency first aid procedures 5. Methods for safe handling and disposal 6. Primary routes of entry 7. Exposure limits and carcinogenic potential Radioactive Hazards A Radioactivity may be encountered in the clinical laboratory when procedures using radioisotopes are performed. The amount of radioactivity present in the clinical laboratory is very small and represents little danger; however, the effects of radiation are cumulative related to the amount of exposure. The amount of radiation exposure is related to a combination of time, distance, and shielding. Persons working in a radioactive environment are required to wear measuring devices to determine the amount of radiation they are accumulating. Laboratory personnel should be familiar with the radioactive hazard symbol shown here. This symbol must be displayed on the doors of all areas where radioactive material is present. Exposure to radiation during pregnancy presents a danger to the fetus; personnel who are pregnant or think they may be should avoid areas with this symbol. Electrical Hazards B Figure 1–4 Chemical safety aids. A, emergency shower; B, eye wash station. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, FA Davis, Philadelphia, 2011, with permission.) The laboratory setting contains a large amount of electrical equipment with which workers have frequent contact. The same general rules of electrical safety observed outside the workplace apply. The danger of water or fluid coming in contact with equipment is greater in the laboratory setting. Equipment should not be operated with wet hands. Designated hospital personnel monitor electrical equipment closely; however, laboratory personnel should continually observe for any dangerous conditions, such as frayed cords and overloaded circuits, and report them to the supervisor. Equipment that has become wet should be unplugged and allowed to dry completely before reusing. Equipment also should be unplugged before cleaning. All electrical equipment must be grounded with three-pronged plugs. When an accident involving electrical shock occurs, the electrical source must be removed immediately. This must be done without touching the person or the equipment involved to avoid transferring the current. Turning off the circuit breaker, unplugging the equipment, or moving the equipment using a nonconductive glass or wood object are safe procedures to follow. The victim should receive immediate medical assistance following 3920_Ch01_002-026 23/01/14 9:20 AM Page 12 12 Part One | Background A B C Figure 1–5 Chemical hazard symbols. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, FA Davis, Philadelphia, 2011, with permission.) discontinuation of the electricity. Cardiopulmonary resuscitation (CPR) may be necessary. Fire/Explosive Hazards The Joint Commission (JC) requires that all health-care institutions post evacuation routes and detailed plans to follow in the event of a fire. Laboratory personnel should be familiar with these procedures. When a fire is discovered, all employees are expected to take the actions in the acronym RACE: Rescue—rescue anyone in immediate danger Alarm—activate the institutional fire alarm system Contain—close all doors to potentially affected areas Extinguish/Evacuate—attempt to extinguish the fire, if possible or evacuate, closing the door As discussed previously, laboratory workers often use potentially volatile or explosive chemicals that require special procedures for handling and storage. Flammable chemicals should be stored in safety cabinets and explosion-proof refrigerators, and cylinders of compressed gas should be located away from heat and securely fastened to a stationary device to prevent accidental capsizing. Fire blankets may be present in the laboratory. Persons with burning clothes should be wrapped in the blanket to smother the flames. The NFPA classifies fires with regard to the type of burning material. It also classifies the type of fire extinguisher that is used to control them. This information is summarized in Table 1–2. The multipurpose ABC fire extinguishers are the most common, 3920_Ch01_002-026 23/01/14 9:20 AM Page 13 Chapter 1 | Safety and Quality Assessment HAZARDOUS MATERIALS CLASSIFICATION FIRE HAZARD Flash Point HEALTH HAZARD 4 3 2 1 0 Deadly Extreme Danger Hazardous Slightly Hazardous Normal Material 2 3 SPECIFIC HAZARD Oxidizer Acid Alkali Corrosive Use No Water Radiation 4 3 2 1 0 Below 73 F Below 100 F Below 200 F Above 200 F Will not burn 1 W OXY ACID ALK COR W REACTIVITY 4 May deteriorate 3 Shock and heat may deteriorate 2 Violent chemical change 1 Unstable if heated 0 Stable Figure 1–6 NFPA hazardous material symbols. but the label should always be checked before using. It is important to be able to operate the fire extinguishers. The acronym PASS can be used to remember the steps in the operation: 1. Pull pin 2. Aim at the base of the fire 3. Squeeze handles 4. Sweep nozzle side to side Physical Hazards Physical hazards are not unique to the laboratory, and routine precautions observed outside the workplace apply. General precautions to consider are to avoid running in rooms and hallways, watch for wet Table 1–2 Types of Fires and Fire Extinguishers Fire Type Extinguishing Material Class A Class B Class C Class D Wood, paper, clothing Flammable organic chemicals Electrical Combustible metals Class K Grease, oils, fats 13 floors, bend the knees when lifting heavy objects, keep long hair pulled back, avoid dangling jewelry, and maintain a clean, organized work area. Closed-toed shoes that provide maximum support are essential for safety and comfort. Q UA L I T Y A SSE S SMEN T The term quality assessment (QA) refers to the overall process of guaranteeing quality patient care and is regulated throughout the total testing system. Quality system refers to all of the laboratory’s policies, processes, procedures, and resources needed to achieve quality testing.15 In a clinical laboratory, a quality assessment program includes not only testing controls, referred to as quality control (QC), but also encompasses preexamination variables (e.g., specimen collection, handling, and storage), examination variables (e.g., reagent and test performance, instrument calibration and maintenance, personnel requirements, and technical competence), postexamination variables (e.g., reporting of results and interpretation), and documentation that the program is being meticulously followed. The original terms preanalytical, analytical, and postanalytical have been replaced with the International Organization for Standardization (ISO) standard terms of preexamination, examination, and postexamination. Included in a QA program are procedure manuals, internal quality control, external quality control, electronic quality control, calibration or calibration verification, standardization, proficiency testing (PT), more formally known as external quality assessment (EQA),16 record keeping, equipment maintenance, safety programs, training, education and competency assessment of personnel, and a scheduled and documented review process. Essentially, QA is the continual monitoring of the entire test process from test ordering and specimen collection through reporting and interpreting results. Written policies and documented actions as they relate to the patient, the laboratory, ancillary personnel, and the health-care provider are required. Having written remedial actions mandating the steps to take when any part of the system fails is essential to a QA program. QA in the urinalysis laboratory—or any other laboratory department—is an integration of many factors. This section Type/Composition of Fire Extinguisher Class A Class B Class C None Class ABC Class K Water Dry chemicals, carbon dioxide, foam, or halon Dry chemicals, carbon dioxide, or halon Sand or dry powder Dry chemicals Liquid designed to prevent splashing and cool the fire. From Strasinger, SK and DiLorenzo, MA: The Phlebotomy Textbook, third edition, FA Davis, Philadelphia, 2011, p.73, with permission. 3920_Ch01_002-026 23/01/14 9:20 AM Page 14 14 Part One | Background will provide a collection of the procedures essential for providing quality urinalysis. In the following chapters, the methods of ensuring accurate results will be covered on an individual basis for each of the tests. Documentation of QA procedures is required by all laboratory accreditation agencies, including the Joint Commission (JC), College of American Pathologists (CAP), American Association of Blood Banks (AABB), American Osteopathic Association (AOA), American Society of Histocompatibility and Immunogenetics (ASHI), and the Commission on Laboratory Assessment (COLA); it is also required for Medicare reimbursement. Guidelines published by CAP and the CLSI provide very complete instructions for documentation and are used as a reference for the ensuing discussion of the specific areas of urinalysis QC and QA.16–18 Documentation in the form of a procedure manual is required in all laboratories, and this format is used as the basis for the following discussion. URINALYSIS SECTION SPECIMEN ACCEPTABILITY/LABELING Prepared by: Initial approval: Procedure placed in use: Revised: Reason for revision: Effective Date Supervisor Approval Medical Director Approval Reviewed Reviewed Reviewed Reviewed Urinalysis Procedure Manual A procedure manual containing all the procedures performed in the urinalysis section must be available for reference in the working area and must comply with the CLSI guidelines. The following information is included for each procedure: principle or purpose of the test, clinical significance, patient preparation, specimen type and method of collection, specimen acceptability and criteria for rejection, reagents, standards and controls, instrument calibration and maintenance protocols and schedules, stepby-step procedure, calculations, frequency and tolerance limits for controls and corrective actions, reference values and critical values, interpretation of results, specific procedure notes, limitations of the method, method validation, confirmatory testing, recording of results, references, effective date, author, and review schedule. Current package inserts should be reviewed and available at the workplace. Electronic manuals are acceptable and must be readily available to all personnel. As with written procedural manuals, electronic versions must be subjected to proper document control (i.e., only authorized persons may make changes, changes are dated/signed [manually or electronically], and there is documentation of periodic review).17,18 Evaluating procedures and adopting new methodologies is an ongoing process in the clinical laboratory. Whenever changes are made, the procedure should be reviewed, referenced, and signed by a person with designated authority, such as the laboratory director or section supervisor (Fig. 1–7), and personnel should be notified of the changes. Documentation of an annual review of all procedures by the designated authority must also be substantiated. Preexamination Variables Preexamination variables occur before the actual testing of the specimen and include test requests, patient preparation, timing, specimen collection, handling, and storage. Health-care personnel outside the clinical laboratory control many of these factors, such as ordering tests and specimen collection. Communication Reviewed Figure 1–7 Example of procedure review documentation. (Adapted from the Department of Pathology, St. Joseph Hospital, Omaha, NE.) between departments and adequate training on the correct procedures for ordering a test, collecting, and transporting the specimen improves the turnaround time (TAT) of results, avoids duplication of test orders, and ensures a high-quality specimen. TAT is defined as the amount of time required from the point at which a test is ordered by the health-care provider until the results are reported to the health-care provider. Laboratories determine the TAT for tests including both stat and routine tests as appropriate. The laboratory can then monitor the TATs to determine areas in the process that need improvement. This can be determined by creating a cause-and-effect diagram, as shown in Figure 1–8. Specimen Collection and Handling Specific information on specimen collection and handling should be stated at the beginning of each procedure listed in the manual. Requisition forms and computerized entry forms should designate the type of urine specimen to be collected and the date and time of collection. The form should include space for recording (1) the actual date and time of specimen collection, (2) whether the specimen was refrigerated before transporting, (3) the time the specimen was received in the laboratory and the time the test was performed, (4) tests requested, (5) an area for specific instructions that might affect the results of the analysis, and (6) patient identification information.18 The patient’s sex, age or date of birth, and, when appropriate, the source of the specimen and the time it was collected must be documented.15 Patient preparation (e.g., fasting or elimination of interfering medications), type and volume of specimen required, 3920_Ch01_002-026 23/01/14 9:20 AM Page 15 Chapter 1 | Safety and Quality Assessment CAUSE-AND-EFFECT DIAGRAM URINALYSIS TURN-AROUND-TIME (TAT) Collection Transport Missed collection 15 Delayed delivery Incorrect specimen type Contaminated specimen container Insufficient urine volume Patient misidentification Special conditions not followed Lost label Insufficient container No requisition Leaking closure lid Missed pick-up Specimen misplaced Incorrect storage No chemical preservative Patient preparation Incorrect container Time of collection not indicated Distance Transport staff unavailable Not refrigerated Not fasting Medications Incomplete requisition UA results TAT Interfering substances Instrument malfunction Report not charted Procedure failure Computer down Wrong specimen tested Printer error Failure to send report Inadequate volume Test analysis Reporting Figure 1–8 Cause-and-effect diagram for analyzing urinalysis TAT. and the need for sterile or opaque containers must be included with the specific procedure. All urine specimens should be examined within 2 hours. If this is not possible, written instructions for preserving the specimen must be available.18 Instructions of a general nature, such as procedures for collecting clean-catch and timed specimens, specimen processing, and printed instructions that are given to patients, are also included in the manual. Criteria for specimen rejection for both physical characteristics and labeling errors must be present. In Table 1–3 is Table 1–3 an example of a policy for handling mislabelled specimens. Written criteria for rejecting specimens must be documented and available to the health-care provider and nursing staff.18 Table 1–4 lists the criteria for urine specimen rejection. Laboratory personnel must determine the suitability of a specimen and document any problems and corrective actions taken. An example of an internal laboratory quality improvement form is shown in Figure 1–9. It is used as a tool to document a problem at the point of discovery, describing what happened and the immediate corrective action taken. This Policy for Handling Mislabeled Specimens Do NOT assume any information about the specimen or patient. Do NOT relabel an incorrectly labeled specimen. Do NOT discard the specimen until investigation is complete. Leave specimen EXACTLY as you receive it; put in the refrigerator for preservation until errors can be resolved. Notify floor, nursing station, doctor’s office, etc. of problem and why it must be corrected for analysis to continue. Identify problem on specimen requisition with date, time, and your initials. Make person responsible for specimen collection participate in solution of problem(s). Any action taken should be documented on the requisition slip. Report all mislabeled specimens to the appropriate supervisor. From Schweitzer, SC, Schumann, JL, and Schumann, GB: Quality assurance guidelines for the urinalysis laboratory. Journal of Medical Technology 3(11): 568, 1986, with permission. 3920_Ch01_002-026 23/01/14 9:20 AM Page 16 16 Part One | Background Table 1–4 Criteria for Urine Specimen Rejection Unlabeled containers Nonmatching labels and requisition forms Contaminated specimens with feces or toilet paper Containers with contaminated exteriors Insufficient volume of urine Improperly transported or preserved specimens Delay between time of collection and receipt in the laboratory enables the laboratory director to capture the information to determine the root cause analysis and develop a preventive or corrective action plan. Laboratory information systems have the capability to electronically generate these forms for review. An acceptable specimen requires verification of the patient’s identification information on the requisition form and the container label, timely transport to the laboratory, the presence of refrigeration or recommended preservative if transport was delayed, and collection of an adequate amount of the correct urine specimen type in a noncontaminated, tightly closed container.18 Examination Variables The examination variables are the processes that directly affect the testing of specimens. They include reagents, instrumentation and equipment, testing procedure, QC, preventive maintenance (PM), access to procedure manuals, and competency of personnel performing the tests. Reagents The manual should state the name and chemical formula of each reagent used, instructions for preparation, when necessary, or company source of prepared materials, storage requirements, and procedures for reagent QC. The type of water used for preparing reagents and controls must be specified. Distilled or deionized water or clinical laboratory reagent water (CLRW) must be available. A bold-type statement of any safety or health precautions associated with reagents should be present. An example of this would be the heat produced in the Clinitest reaction. All reagents and reagent strips must be properly labeled with the date of preparation or opening, purchase and received date, expiration date, and appropriate safety information. Reagent strips should be checked against known negative and positive control solutions on each shift or at a minimum once a day, and whenever a new bottle is opened. Reagents are checked daily or when tests requiring their use are requested. Results of all reagent checks are properly recorded. Reagent strips must never be refrigerated, and must be recapped immediately after removing each strip. when limitations or linearity are exceeded, such as dilution procedures, must be clearly stated in the procedure manual. Instructions detailing the appropriate recording procedures must be included. The most frequently encountered instruments in the urinalysis laboratory are refractometers, osmometers, automated reagent strip readers, and automated microscopy instruments. Refractometers are calibrated on each shift against deionized water (1.000) and a known control, such as 5% saline (1.022 ± 0.001) or 9% sucrose (1.034 ± 0.001). Two levels of commercial controls are available for the osmometer, urine reagent strip tests, and hCG kit tests. All control values must be recorded. Automated urinalysis systems and reagent strip readers are calibrated using manufacturer-supplied calibration materials following the protocol specified by the manufacturer. Both positive and negative control values must be run and recorded (Fig. 1–10). Evidence of corrective action for any failed QC tests must be documented. No patient’s testing may be performed until QC is acceptable. Equipment found in the urinalysis laboratory commonly includes refrigerators, centrifuges, microscopes, and water baths. Temperatures of refrigerators and water baths should be taken daily and recorded. Calibration of centrifuges is customarily performed every 3 months, and the appropriate relative centrifugal force for each setting is recorded. Centrifuges are routinely disinfected on a weekly basis. Microscopes should be kept clean at all times and have an annual professional cleaning. A routine PM schedule for instruments and equipment should be prepared as mandated by the JC or CAP guidelines, and records kept of all routine and nonroutine maintenance performed. Deionized water used for reagent preparation is quality controlled by checking pH and purity meter resistance on a weekly basis and the bacterial count on a monthly schedule. All results must be recorded on the appropriate forms. Testing Procedure Detailed, concise testing instructions are written in a stepby-step manner. Instructions should begin with specimen preparation, such as time and speed of centrifugation, and include types of glassware needed, time limitations and stability of specimens and reagents, calculation formulas and a sample calculation, health and safety precautions, and procedures. Additional procedure information including reasons for special precautions, sources of error and interfering substances, helpful hints, clinical situations that influence the test, alternative procedures, and acceptable TATs for stat tests are listed under the title of Procedure Notes following the step-by-step procedure. Reference sources should be listed. Manufacturer’s package inserts may be included but cannot replace the written procedure. The laboratory director must sign and date new procedures and all modifications of procedures before they are used.15 Instrumentation and Equipment Quality Control Instructions regarding the operation, performance and frequency of calibration, limitations, and procedures to follow Quality control refers to the materials, procedures, and techniques that monitor the accuracy, precision, and reliability 3920_Ch01_002-026 23/01/14 9:20 AM Page 17 Chapter 1 | Safety and Quality Assessment 17 Quality Improvement Follow-up Report CONFIDENTIAL Instructions: Section I should be completed by the individual identifying the event. Date of report: Date of incident: Patient MR# Reported by: Date/time of discovery: Patient accession# Section I Summary of incident — describe what happened What immediate corrective action was taken? Provide the ORIGINAL to team leader/technical specialist within 24 hours of incident discovery Date: To: Forwarded for follow-up: Date: To: Section II. Management investigation: Tracking # Instructions: Section II should be completed by laboratory management within 72 hours Check appropriate problem category Unacceptable patient samples (Due to hemolysis, QNS, or contaminated) Wrong tube type Equipment-related event Misidentified sample Standard operating procedure deviation Wrong location Communication problem/complaint Other (explain) Accident Explain answers: Preventive/corrective action recommendations: Technical specialist/team leader: Date: Medical director review: Date: Quality assurance review: Date: FDA reportable: Yes or no Date reported: Figure 1–9 Sample of Quality Improvement Follow-up Report form. (From Danville Regional Medical Center Laboratory, Danville, VA, with permission.) 3920_Ch01_002-026 23/01/14 9:20 AM Page 18 18 Part One | Background QUALITY CONTROL GLU mg/dL BIL Month:__________20_____ Positive control SP BLD PH PROT NIT GR mg/dL 1.0 mg/dL KET Negative control LEU GLU BIL KET SP BLD PH PROT NIT EST GR mg/dL mg/dL 1.0 mg/dL LEU EST Control values Reagent Lot # EXP TECH DATE Figure 1–10 Sample instrument QC recording sheet. (Adapted from the Department of Pathology, Methodist Hospital, Omaha, NE, with permission.) of a laboratory test. QC procedures are performed to ensure that acceptable standards are met during the process of patient testing. Specific QC information regarding the type of control specimen preparation and handling, frequency of use, tolerance levels, and methods of recording should be included in the step-by-step instructions for each test. QC is performed at scheduled times, such as at the beginning of each shift or before testing patient samples, and it must always be performed if reagents are changed, an instrument malfunction has occurred, or if test results are questioned by the health-care provider. Control results must be recorded in a log, either paper or electronic. Patient test results may not be reported until the QC is verified. Both external quality control monitoring and internal and electronic quality control processes are practiced in the urinalysis laboratory. External Quality Control External quality controls are used to verify the accuracy (ability to obtain the expected result) and precision (ability to obtain the same result on the same specimen) of a test and are exposed to the same conditions as the patient samples. Reliability is the ability to maintain both precision and accuracy. Commercial controls are available for the urine chemistry tests, specific gravity, and for certain microscopic constituents. Analysis of two levels of control material is required. The concentration of controls should be at medically significant levels and should be as much like the human specimen as possible. Documentation of QC includes dating and initialling the material when it is first opened and recording the manufacturer’s lot number and the expiration date each time a control is run and the test result is obtained. Food and Drug Administration (FDA) standards require that control material test negative for HIV and hepatitis B virus. External controls are tested and interpreted in the laboratory by the same person performing the patient testing. Control data are evaluated before releasing patient results. Data obtained from repeated measurements have a Gaussian distribution or spread in the values that indicate the ability to repeat the analysis and obtain the same value. The laboratory, after repeated testing, establishes the value for each analyte, and the mean and standard deviation is calculated. The control mean is the average of all data points and the standard deviation (SD) is a measurement statistic that describes the average distance each data point in a normal distribution is from the mean. The coefficient of variation (CV) is the SD expressed as a percentage of the mean. The CV indicates whether the distribution of values about the mean is in a narrow versus broad range and should be less than 5%. Confidence intervals are the limits between which the specified proportion or percentage of results will lie. Control ranges are determined by setting confidence limits that are within ±2 SD or ±3 SD of the mean, which indicates that 95.5% to 99.7% of the values are expected to be within that range. Values are plotted on Levy-Jennings control charts to visually monitor control values. Immediate decisions about patient results are based on the ability of control values to remain within a preestablished limit. Changes in accuracy of results are indicated by either a trend that is a gradual changing in the mean in one direction or a shift that is an abrupt change in the mean (Fig. 1–11). Changes in precision are shown by a large amount of scatter about the mean and an uneven distribution above and below the mean that are most often caused by errors in technique. Corrective action, including the use of new reagents, reagent strips, or controls, and the verification of lot numbers and expiration dates, must be taken when control values are outside the tolerance limits. All corrective actions taken are documented. A protocol for corrective action is shown in Figure 1–12. A designated supervisor reviews all QC results. Laboratories may participate in a commercial QC program. Results from the same lot of QC material sent by the manufacturer to participating laboratories are returned to the manufacturer for statistical analysis and comparison with other laboratories using the same methodology. Internal Quality Control Internal quality control consists of internal monitoring systems built in to the test system and are called internal or procedural controls.16,19 Internal controls monitor the sufficient addition of a patient specimen or reagent, the instruments/reagents 3920_Ch01_002-026 23/01/14 9:20 AM Page 19 Chapter 1 | Safety and Quality Assessment 2 4 6 8 Day 10 12 19 A. Record all actions taken and the resolution of any problems 14 16 18 B. Use the flow diagram below: 20 + 2 S.D. + 1 S.D. X – 1 S.D. – 2 S.D. In Control + 2 S.D. + 1 S.D. X – 1 S.D. – 2 S.D. Shift 1. Run control In control Out of control Proceed with testing Go to Step 2 2. Inspect control for: Outdate (age), proper storage, correct lot number, signs of contamination Yes there is a problem No obvious explanation Make new control and retest Retest + 2 S.D. + 1 S.D. X – 1 S.D. – 2 S.D. Trend Figure 1–11 Levy-Jennings charts showing in-control, shift, and trend results. interaction, and, for lateral flow test methods, whether the sample migrated through the test strip properly.16 Electronic Controls External quality control (EQC) uses a mechanical or electrical device in place of a liquid QC specimen. This type of QC can be internal or an external component inserted into a point of care (POC) instrument. EQC verifies the functional ability of a testing device, but it does not verify the integrity of the testing supplies. Many test systems use a combination of external and internal controls to verify that the entire test system is working properly. Proficiency Testing (External Quality Assessment) PT or EQA is the testing of unknown samples received from an outside agency, and provides unbiased validation of the quality of patient test results. Several commercial vendors provide proficiency testing such as CAP. Laboratories subscribing to these programs receive lyophilized or ready-to-use specimens for routine urinalysis and Kodachromes or color plates for sediment constituent identification. The results are returned to the proficiency testing vendors, where they are statistically analyzed with those from all participating laboratories, and a report is returned to the laboratory director. The laboratory accuracy is evaluated and compared with other laboratories using the same method of analysis. Corrective action must be taken for unacceptable results.16 The Clinical Laboratory Improvement Amendments (CLIA) mandates comparison testing for laboratory accreditation.19 Personnel and Facilities Quality control is only as good as the personnel performing and monitoring it. Personnel assessment includes education In control Out of control Proceed with testing Go to Step 3 3. Make up new bottle of control In control Out of control Discard old control and proceed with testing Go to Step 4 4. Open new can of reagent strips and test with new control In control Out of control Discard bad reagent strips and proceed with testing Switch lot numbers and retest In control Discard entire lot, notify manufacturer, and proceed with testing Out of control Notify supervisor or resource person: do not proceed with testing Figure 1–12 “Out-of-control” procedures. (From Schweitzer, SC, Schumann, JL, and Schumann, GB: Quality assurance guidelines for the urinalysis laboratory. Journal of Medical Technology 3(11):567–572, 1986, with permission.) and training, continuing education, competency assessment, and performance appraisals. Each new employee must have documentation of training during orientation to the laboratory. This is a checklist of procedures and must include date and initials of the person doing the training and of the employee being trained. Up-to-date reference materials and atlases should be readily available, and documentation of continuing education must be maintained.16 An adequate, uncluttered, safe working area is also essential for both quality work and personnel morale. Standard precautions for handling body fluids must be followed at all times. 3920_Ch01_002-026 23/01/14 9:20 AM Page 20 20 Part One | Background Postexamination Variables Postexamination variables are processes that affect the reporting of results and correct interpretation of data. Reporting Results Standardized reporting formats and, when applicable, reference ranges should be included with each procedure covered in the procedure manual. A written procedure for reporting, reviewing, and correcting errors must be present. Forms for reporting results should provide adequate space for writing and should present the information in a logical sequence. Standardized reporting methods minimize health-care provider confusion when interpreting results (Fig. 1–13). Electronic transmission is now the most common method for reporting results. Many urinalysis instruments have the capability for the operator to transmit results directly from the instrument to the designated health-care provider. It is essential that the operator carefully review results before transmittal. Results may also be manually entered into the laboratory computer system and then transmitted to the health-care providers. MICROSCOPIC QUANTITATIONS Quantitate an average of 10 representative fields. Do not quantitate budding yeast, mycelial elements, Trichomonas, or sperm, but do note their presence with the appropriate LIS code. Epithelial cells/LPF None: Rare: Few: Moderate: Many: Casts/LPF None: Numerical ranges: RBCs/HPF None: Numerical ranges: WBCs/HPF None: Numerical ranges: Crystals/HPF None: Rare: Few: Moderate: Many: Bacteria/HPF None: Rare: Few: Moderate: Many: Mucous threads Rare: Few: Moderate: Many: 0 0–5 5–20 20–100 >100 0 0–2, 2–5, 5–10, >10 0 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 0 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 0 0–2 2–5 5–20 >20 0 0–10 10–50 50–200 >200 0–1 1–3 3–10 >10 Figure 1–13 Sample standardized urine microscopic reporting format. (From University of Nebraska Medical Center, Omaha, NE, with permission.) Errors may be discovered in the laboratory through a QA procedure known as the delta check that compares a patient’s test results with the previous results. Variation outside the established parameters alerts laboratory personnel to the possibility of an error that occurred during the testing procedure or in patient identification. Autoverification is often programmed into many laboratory analyzers.4 Erroneous results must be corrected in a timely manner to assure that the patient does not receive treatment based on incorrect results. Errors can occur in patient identification, specimen labeling, or result transcription. The patient’s record should be corrected as soon as the error is detected; however, the original result must not be erased in the event that the health-care provider treated the patient based on the erroneous results. Appropriate documentation of erroneous results should follow institutional protocol. The telephone is frequently used to transmit results of stat tests and critical values. Calls requesting additional results may be received from personnel on hospital units and from healthcare providers. When telephoning results, confirm that the results are being reported to the appropriate person. The time of the call and the name of the person receiving the results must be documented according to the facility’s policy. The Joint Commission Patient Safety Goals require that when verbally reporting test results the information must be repeated by the person receiving the information and documented by the person giving the report. Written procedures should be available for the reporting of critical values (Fig. 1–14). In laboratories analyzing pediatric Reporting Critical Results In Urinalysis POSITIVE KETONES For children age 2 years and younger: All results positive for ketones should be telephoned to the appropriate nursing unit. Document the following information in the computer as a chartable footnote appended to the result: Time of telephone call Initials of the person making the call Name of the person receiving the telephone call POSITIVE CLINITEST For children age 2 years and younger: All Clinitest results should be telephoned to the appropriate nursing unit. Document the following information in the computer as a chartable footnote appended to the result: Time of telephone call Initials of the person making the call Name of the person receiving the telephone call Figure 1–14 An example of procedure instructions for reporting critical values in the urinalysis section. A procedure review document similar to that shown in Figure 1–7 would accompany this instruction sheet. 3920_Ch01_002-026 23/01/14 9:20 AM Page 21 Chapter 1 | Safety and Quality Assessment SUMMARY 1-1 Quality Assessment Errors Preexamination Patient misidentification Wrong test ordered Incorrect urine specimen type collected Insufficient urine volume Delayed transport of urine to the laboratory Incorrect storage or preservation of urine Examination Sample misidentification Erroneous instrument calibration Reagent deterioration Poor testing technique Instrument malfunction Interfering substances present Misinterpretation of quality control data Postexamination Patient misidentification Poor handwriting Transcription error Poor quality of instrument printer Failure to send report Failure to call critical values Inability to identify interfering substances specimens, this should include the presence of ketones or reducing substances in newborns. Interpreting Results The specificity and the sensitivity for each test should be included in the procedure manual for correct interpretation of results. Sensitivity and specificity vary among manufacturers. All known interfering substances should be listed for evaluation of patient test data. A well-documented QA program ensures quality test results and patient care. Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Clinical and Laboratory Standards Institute: Protection of laboratory workers from occupationally acquired infections: approved guideline, ed 3, CLSI Document M29-A3, Clinical and Laboratory Standards Institute, Wayne, PA, 2005, CLSI. 2. Clinical and Laboratory Standards Institute: Clinical laboratory safety: approved guideline, ed 2, CLSI Document GP17-A2, Clinical and Laboratory Standards Institute, Wayne, PA, 2004, CLSI. 21 3. Clinical and Laboratory Standards Institute: Clinical laboratory waste management: approved guideline, ed 2, CLSI Document GP05-A3, Clinical and Laboratory Standards Institute, Wayne, PA, 2005, CLSI. 4. Strasinger SK, Di Lorenzo MS, The Phlebotomy Textbook, ed 3, 2011. F.A. Davis, Philadelphia. 5. Centers for Disease Control and Prevention. Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings, 2007. www.cdc.gov/hicpac/ 2007isolationPrecations.html. 6. Occupational Exposure to Blood-Borne Pathogens, Final Rule. Federal Register 29 (Dec 6), 1991. 7. Occupation Safety and Health Administration. Revision to OSHA’s Bloodborne Pathogens Standard. 2001. Department of Labor. Web site: www.osha.gov/SLTC/bloodbornepathogens. 8. Centers for Disease Control and Prevention. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV and HIV and Recommendations for Post-exposure Prophylaxis. MMWR June 29, 2001: 50(RR11); 1–42. Web site: www.cdc.gov 9. Centers for Disease Control and Prevention. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposure to HIV and Recommendations for Postexposure Prophylaxis. MMWR September 17, 2005: 54(RR09); 1–17. Web site: www.cdc.gov 10. NIOSH Alert. Preventing Allergic Reactions to Natural Rubber Latex in the Workplace. DHHS (NIOSH) Publication 97-135. National Institute for Occupational Safety and Health, Cincinnati, OH, 1997. 11. Centers for Disease Control and Prevention. Guideline for Hand Hygiene in Health-Care Settings: Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force, MMWR 2002:51(RR16). [Inclusive page numbers] 12. Centers for Disease Control and Prevention. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. Web site: www.cdc.gov/hicpac/pdf/guidelines/Disinfection_ Nov_2008.pdf 13. Occupational Exposure to Hazardous Chemicals in Laboratories, Final Rule. Federal Register 55 (Jan 31), 1990. 14. National Fire Protection Association: Hazardous Chemical Data, No. 49. Boston, NFPA, 1991. 15. Centers for Medicare & Medicaid Services, Department of Health and Human Services: Clinical Laboratory Improvement Amendments, Updated Regulations, Brochure #1, How do they affect my laboratory? www.cms.hhs.gov/CLIA/05_CLIA_ Brochures.asp Accessed November 2006. 16. Clinical and Laboratory Standards Institute (CLSI): Quality Practices in Noninstrumented Point of Care Testing: An Instructional Manual and Resources for Health Care Workers. Approved Guideline. CLSI document POCT08-A, Wayne, PA, 2010. 17. College of American Pathologists: Commission on Laboratory Accreditation, Urinalysis Checklist. College of American Pathologists, Skokie, IL, 2005. 18. Clinical and Laboratory Standards Institute (CLSI), Urinalysis: Approved Guideline-Third Edition, CLSI document GP16-A3, 2009. 19. Centers for Medicare & Medicaid Services, Department of Health and Human Services: Clinical Laboratory Improvement Amendments, Brochure #4: Equivalent Quality Control Procedures. www.cms.hhs.gov/CLIA/05_CLIA_Brochures.asp Accessed November 2006. 3920_Ch01_002-026 23/01/14 9:20 AM Page 22 22 Part One | Background Study Questions 1. In the urinalysis laboratory the primary source in the chain of infection would be: A. Patients B. Needlesticks C. Specimens D. Biohazardous waste 2. The best way to break the chain of infection is: A. Hand sanitizing B. Personal protective equipment C. Aerosol prevention D. Decontamination 3. The current routine infection control policy developed by CDC and followed in all health-care settings is: A. Universal Precautions B. Isolation Precautions C. Blood and Body Fluid Precautions D. Standard Precautions 4. An employee who is accidentally exposed to a possible blood-borne pathogen should immediately: A. Report to a supervisor B. Flush the area with water C. Clean the area with disinfectant D. Receive HIV prophylaxis 5. Personnel in the urinalysis laboratory should wear lab coats that: A. Do not have buttons B. Are fluid-resistant C. Have short sleeves D. Have full-length zippers 6. All of the following should be discarded in biohazardous waste containers except: A. Urine specimen containers B. Towels used for decontamination C. Disposable lab coats D. Blood collection tubes 7. An employer who fails to provide sufficient gloves for the employees may be fined by the: A. CDC B. NFPA C. OSHA D. FDA 8. An acceptable disinfectant for blood and body fluid decontamination is: A. Sodium hydroxide B. Antimicrobial soap C. Hydrogen peroxide D. Sodium hypochlorite 9. Proper handwashing includes all of the following except: A. Using warm water B. Rubbing to create a lather C. Rinsing hands in a downward position D. Turning on the water with a paper towel 10. Centrifuging an uncapped specimen may produce a biologic hazard in the form of: A. Vectors B. Sharps contamination C. Aerosols D. Specimen contamination 11. An employee who accidentally spills acid on his arm should immediately: A. Neutralize the acid with a base B. Hold the arm under running water for 15 minutes C. Consult the MSDSs D. Wrap the arm in gauze and go to the emergency department 12. When combining acid and water, ensure that: A. Acid is added to water B. Water is added to acid C. They are added simultaneously D. Water is slowly added to acid 13. An employee can learn the carcinogenic potential of potassium chloride by consulting the: A. Chemical hygiene plan B. Material safety data sheets C. OSHA standards D. Urinalysis procedure manual 14. Employees should not work with radioisotopes if they are: A. Wearing contact lenses B. Allergic to iodine C. Sensitive to latex D. Pregnant 3920_Ch01_002-026 23/01/14 9:20 AM Page 23 Chapter 1 | Safety and Quality Assessment 23 15. All of the following are safe to do when removing the source of an electric shock except: A. Pulling the person away from the instrument B. Turning off the circuit breaker C. Using a glass container to move the instrument D. Unplugging the instrument 23. The classification of a fire that can be extinguished with water is: A. Class A B. Class B C. Class C D. Class D 16. The acronym PASS refers to: A. Presence of vital chemicals B. Operation of a fire extinguisher C. Labeling of hazardous material D. Presence of radioactive substances 24. Employers are required to provide free immunization for: A. HIV B. HTLV-1 C. HBV D. HCV 17. The system used by firefighters when a fire occurs in the laboratory is: A. MSDS B. RACE C. NFPA D. PASS 25. A possible physical hazard in the hospital is: A. Wearing closed-toed shoes B. Not wearing jewelry C. Having short hair D. Running to answer the telephone 18. A class ABC fire extinguisher contains: A. Sand B. Water C. Dry chemicals D. Acid 26. Quality assessment refers to: A. Analysis of testing controls B. Increased productivity C. Precise control results D. Quality of specimens and patient care 19. The first thing to do when a fire is discovered is to: A. Rescue persons in danger B. Activate the alarm system C. Close doors to other areas D. Extinguish the fire if possible 27. During laboratory accreditation inspections, procedure manuals are examined for the presence of: A. Critical values B. Procedure references C. Procedures for specimen preservation D. All of the above 20. If a red rash is observed after removing gloves, the employee: A. May be washing her hands too often B. May have developed a latex allergy C. Should apply cortisone cream D. Should not rub the hands so vigorously 28. As supervisor of the urinalysis laboratory, you have just adopted a new procedure. You should: A. Put the package insert in the procedure manual B. Put a complete, referenced procedure in the manual C. Notify the microbiology department D. Put a cost analysis study in the procedure manual 21. Pipetting by mouth is: A. Acceptable for urine but not serum B. Not acceptable without proper training C. Acceptable for reagents but not specimens D. Not acceptable in the laboratory 29. Indicate whether each of the following would be considered a 1) preexamination, 2) examination, or 3) postexamination factor by placing the appropriate number in the blank: _____ Reagent expiration date _____ Rejecting a contaminated specimen _____ Constructing a Levy-Jennings chart _____ Telephoning a positive Clinitest result on a newborn _____ Calibrating the centrifuge _____ Collecting a timed urine specimen 22. The NPFA classification symbol contains information on all of the following except: A. Fire hazards B. Biohazards C. Reactivity D. Health hazards 3920_Ch01_002-026 23/01/14 9:20 AM Page 24 24 Part One | Background 30. The testing of sample from an outside agency and the comparison of results with participating laboratories is called: A. External QC B. Electronic QC C. Internal QC D. Proficiency testing 33. When a new bottle of QC material is opened, what information is placed on the label? A. The supervisor’s initials B. The lot number C. The date and the laboratory worker’s initials D. The time the bottle was opened 31. A color change that indicates that a sufficient amount of patient’s specimen or reagent is added correctly to the test system would be an example of: A. External QC B. Equivalent QC C. Internal QC D. Proficiency testing 34. When a control is run, what information is documented? A. The lot number B. Expiration date of the control C. The test results D. All of the above 32. What steps are taken when the results of reagent strip QC are outside of the stated confidence limits? A. Check the expiration date of the reagent strip B. Run a new control C. Open a new reagent strips container D. All of the above Case Studies and Clinical Situations 1. State a possible reason for an accreditation team to report a deficiency in the following situations: a. The urine microscopic reporting procedure has been recently revised. b. An unusually high number of urine specimens are being rejected because of improper collection. c. A key statement is missing from the Clinitest procedure. d. Open control bottles in the refrigerator are examined. 2. As the new supervisor of the urinalysis section, you encounter the following situations. Explain whether you would accept them or take corrective action. a. You are told that the supervisor always performs the CAP proficiency survey. b. QC is not performed daily on the reagent strips. c. The urinalysis section is primarily staffed by personnel assigned to other departments for whom you have no personnel data. 3. The medical technologist was assigned to test 10 urine specimens chemically. She removed 10 strips from the container and proceeded with testing. Several patients’ urine indicated a trace positive glucose in the urine. She then opened a new bottle of reagent dipsticks and proceeded to perform the QC. The negative control also tested as a trace positive for glucose. The medical technologist consulted the supervisor. The supervisor ran the QC and the results were in the correct range. After observing the medical technologist’s technique, the supervisor realized that the medical technologist was waiting too long to read the glucose results and therefore reporting erroneous results. a. What is wrong with this scenario? b. Who should run the QC for each test? Why? c. When should controls be run? d. What do you do when the QC is out of range? e. When can you report patient results? 3920_Ch01_002-026 23/01/14 9:20 AM Page 25 Chapter 1 | Safety and Quality Assessment 4. An outpatient urine specimen was delivered to the laboratory at 0800 and placed on the counter in the Urinalysis department. The medical laboratory scientist performed urinalysis on the specimen at 1130. The following results were abnormal: Clarity: Cloudy pH: 9.0 Nitrite: Positive The patient was a known diabetic; however, the glucose result was negative. a. What could be a possible cause for the abnormal results? 25 b. Where would the information concerning what should have been done with this specimen be found and the criteria for rejection? c. What QA procedure may have detected this error? d. What form will need to be completed for this scenario? e. How might this affect this patient’s care? f. How will the corrected results be documented? 3920_Ch01_002-026 23/01/14 9:20 AM Page 26 3920_Ch02_027-038 23/01/14 10:08 AM Page 27 CHAPTER 2 Introduction to Urinalysis LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 2-1 List three major organic and three major inorganic chemical constituents of urine. 2-8 Discuss the actions of bacteria on an unpreserved urine specimen. 2-2 Describe a method for determining whether a questionable fluid is urine. 2-9 Briefly discuss five methods for preserving urine specimens, including their advantages and disadvantages. 2-3 Recognize normal and abnormal daily urine volumes. 2-4 Describe the characteristics of the recommended urine specimen containers. 2-5 Describe the correct methodology for labeling urine specimens. 2-6 State four possible reasons a laboratory would reject a urine specimen. 2-7 List 10 changes that may take place in a urine specimen that remains at room temperature for more than 2 hours. 2-10 Instruct a patient in the correct procedure for collecting the following specimens: random, first morning, 24-hour timed, catheterized, midstream clean-catch, suprapubic aspiration, three-glass collection, and pediatric. Identify a diagnostic use for each collection technique. 2-11 Describe the type of specimen needed for optimal results when a specific urinalysis procedure is requested. KEY TERMS Albuminuria First morning specimen Polyuria Anuria Midstream clean-catch specimen Suprapubic aspiration Catheterized specimen Nocturia Timed specimen Chain of custody (COC) Oliguria 3920_Ch02_027-038 23/01/14 10:08 AM Page 28 28 Part One | Background History and Importance Analyzing urine was actually the beginning of laboratory medicine. References to the study of urine can be found in the drawings of cavemen and in Egyptian hieroglyphics, such as the Edwin Smith Surgical Papyrus. Pictures of early physicians commonly showed them examining a bladder-shaped flask of urine (Fig. 2–1). Often these physicians never saw the patient, only the patient’s urine. Although these physicians lacked the sophisticated testing mechanisms now available, they were able to obtain diagnostic information from such basic observations as color, turbidity, odor, volume, viscosity, and even sweetness (by noting that certain specimens attracted ants or tasted sweet). These same urine characteristics are still reported by laboratory personnel. However, modern urinalysis has expanded beyond physical examination of urine to include chemical analysis and microscopic examination of urinary sediment. Many well-known names in the history of medicine are associated with the study of urine, including Hippocrates, who, in the 5th century BCE, wrote a book on “uroscopy.” During the Middle Ages, physicians concentrated their efforts very intensively on the art of uroscopy, receiving instruction in urine examination as part of their training (Fig. 2–2). By 1140 CE, color charts had been developed that described the significance of 20 different colors (Fig. 2–3). Chemical testing progressed from “ant testing” and “taste testing” for glucose to Frederik Dekkers’ discovery in 1694 of albuminuria by boiling urine.1 Figure 2–2 Instruction in urine examination. (Courtesy of National Library of Medicine.) Figure 2–1 Physician examines urine flask. (Courtesy of National Library of Medicine.) The credibility of urinalysis became compromised when charlatans without medical credentials began offering their predictions to the public for a healthy fee. These charlatans, called “pisse prophets,” became the subject of a book published by Thomas Bryant in 1627. The revelations in this book inspired the passing of the first medical licensure laws in England— another contribution of urinalysis to the field of medicine. The invention of the microscope in the 17th century led to the examination of urinary sediment and to the development by Thomas Addis of methods for quantitating the microscopic sediment. Richard Bright introduced the concept of urinalysis as part of a doctor’s routine patient examination in 1827. By the 1930s, however, the number and complexity of the tests performed in a urinalysis had reached a point of impracticality, and urinalysis began to disappear from routine examinations. Fortunately, development of modern testing techniques rescued routine urinalysis, which has remained an integral part of the patient examination. Two unique characteristics of a urine specimen account for this continued popularity: 1. Urine is a readily available and easily collected specimen. 3920_Ch02_027-038 23/01/14 10:08 AM Page 29 Chapter 2 | Introduction to Urinalysis 29 Urea, a metabolic waste product produced in the liver from the breakdown of protein and amino acids, accounts for nearly half of the total dissolved solids in urine. Other organic substances include primarily creatinine and uric acid. The major inorganic solid dissolved in urine is chloride, followed by sodium and potassium. Small or trace amounts of many additional inorganic chemicals are also present in urine (Table 2–1). Dietary intake greatly influences the concentrations of these inorganic compounds, making it difficult to establish normal levels. Other substances found in urine include hormones, vitamins, and medications. Although not a part of the original plasma filtrate, the urine also may contain formed elements, such as cells, casts, crystals, mucus, and bacteria. Increased amounts of these formed elements are often indicative of disease. Urine Volume Urine volume depends on the amount of water that the kidneys excrete. Water is a major body constituent; therefore, the amount excreted is usually determined by the body’s state of Figure 2–3 A chart used for urine analysis. (Courtesy of National Library of Medicine.) 2. Urine contains information, which can be obtained by inexpensive laboratory tests, about many of the body’s major metabolic functions. These characteristics fit in well with the current trends toward preventive medicine and lower medical costs. In fact, the Clinical and Laboratory Standards Institute (CLSI) defines urinalysis as “the testing of urine with procedures commonly performed in an expeditious, reliable, accurate, safe, and cost-effective manner.” Reasons for performing urinalysis identified by CLSI include aiding in the diagnosis of disease, screening asymptomatic populations for undetected disorders, and monitoring the progress of disease and the effectiveness of therapy.2 Urine Formation The kidneys continuously form urine as an ultrafiltrate of plasma. Reabsorption of water and filtered substances essential to body function converts approximately 170,000 mL of filtered plasma to the average daily urine output of 1200 mL. (Refer to Chapter 3.) Urine Composition In general, urine consists of urea and other organic and inorganic chemicals dissolved in water. Urine is normally 95% water and 5% solutes, although considerable variations in the concentrations of these solutes can occur owing to the influence of factors such as dietary intake, physical activity, body metabolism, and endocrine functions. TECHNICAL TIP Should it be necessary to determine whether a particular fluid is urine, the specimen can be tested for its urea and creatinine content. Because both these substances are present in much higher concentrations in urine than in other body fluids, a high urea and creatinine content can identify a fluid as urine. Table 2–1 Primary Components in Normal Urine3 Component Comment Urea Primary organic component. Product of protein and amino acid metabolism Product of creatine metabolism by muscles Product of nucleic acid breakdown in food and cells Primary inorganic component. Found in combination with sodium (table salt) and many other inorganic substances Primarily from salt, varies by intake Combined with chloride and other salts Combines with sodium to buffer the blood Regulates blood and tissue fluid acidity Combines with chloride, sulfate, and phosphate Creatinine Uric acid Chloride Sodium Potassium Phosphate Ammonium Calcium 3920_Ch02_027-038 23/01/14 10:08 AM Page 30 30 Part One | Background hydration. Factors that influence urine volume include fluid intake, fluid loss from nonrenal sources, variations in the secretion of antidiuretic hormone, and need to excrete increased amounts of dissolved solids, such as glucose or salts. Taking these factors into consideration, although the normal daily urine output is usually 1200 to 1500 mL, a range of 600 to 2000 mL is considered normal. Oliguria, a decrease in urine output (which is less than 1 mL/kg/hr in infants, less than 0.5 mL/kg/hr in children, and less than 400 mL/day in adults), is commonly seen when the body enters a state of dehydration as a result of excessive water loss from vomiting, diarrhea, perspiration, or severe burns. Oliguria leading to anuria, cessation of urine flow, may result from any serious damage to the kidneys or from a decrease in the flow of blood to the kidneys. The kidneys excrete two to three times more urine during the day than during the night. An increase in the nocturnal excretion of urine is termed nocturia. Polyuria, an increase in daily urine volume (greater than 2.5 L/day in adults and 2.5 to 3 mL/kg/day in children), is often associated with diabetes mellitus and diabetes insipidus; however, it may be artificially induced by diuretics, caffeine, or alcohol, all of which suppress the secretion of antidiuretic hormone. Diabetes mellitus and diabetes insipidus produce polyuria for different reasons, and analysis of the urine is an important step in the differential diagnosis (Fig. 2–4). Diabetes mellitus is caused by a defect either in the pancreatic production of insulin or in the function of insulin, which results in an increased body glucose concentration. The kidneys do not reabsorb excess glucose, necessitating excretion of increased amounts of water to remove the dissolved glucose from the body. Although appearing to be dilute, a urine specimen from a patient with Polydipsia Polyuria Specific gravity Decreased SG Increased SG Decreased production or Function of ADH Decreased insulin or Decreased function of insulin Diabetes insipidus Increased glucose Diabetes mellitus Figure 2–4 Differentiation between diabetes mellitus and diabetes insipidus. diabetes mellitus has a high specific gravity because of the increased glucose content. Diabetes insipidus results from a decrease in the production or function of antidiuretic hormone (ADH); thus, the water necessary for adequate body hydration is not reabsorbed from the plasma filtrate. In this condition, the urine is truly dilute and has a low specific gravity. Fluid loss in both diseases is compensated by increased ingestion of water (polydipsia), producing an even greater urine volume. Polyuria accompanied by increased fluid intake is often the first symptom of either disease. Specimen Collection As discussed in Chapter 1, urine is a biohazardous substance that requires the observance of Standard Precautions. Gloves should be worn at all times when in contact with the specimen. Containers Specimens must be collected in clean, dry, leak-proof containers. Disposable containers should be used because they eliminate the chance of contamination owing to improper washing. These disposable containers are available in a variety of sizes and shapes, including bags with adhesive for the collection of pediatric specimens and large containers for 24-hour specimens. Properly applied screw-top lids are less likely to leak than are snap-on lids. Containers for routine urinalysis should have a wide mouth to facilitate collections from female patients and a wide, flat bottom to prevent overturning. They should be made of a clear material to allow for determination of color and clarity. The recommended capacity of the container is 50 mL, which allows 12 mL of specimen needed for microscopic analysis, additional specimen for repeat analysis, and enough room for the specimen to be mixed by swirling the container. Individually packaged sterile containers with secure closures should be used for microbiologic urine studies. Sterile containers are also suggested if more than 2 hours elapse between specimen collection and analysis.2 Specially designed sterile containers are available that have a lid with a transfer device that can be assessed with a device called a transfer straw. The transfer straw has a needle and an evacuated tube holder. Urine can be sterilely transferred to tubes containing preservatives for microbiology testing and tubes with conical bottoms for sediment analysis or round bottoms for automated reagent strip testing.4 Additional information and pictures can be found at http//www. bd.com/ds/productCenter/. Labels All specimens must be labeled properly with the patient’s name and identification number, the date and time of collection, and additional information such as the patient’s age and location and the healthcare provider’s name, as required by institutional protocol. Labels must be attached to the container, not to the 3920_Ch02_027-038 23/01/14 10:08 AM Page 31 Chapter 2 | Introduction to Urinalysis 31 Specimen Handling lid, and should not become detached if the container is refrigerated or frozen. Requisitions A requisition form (manual or computerized) must accompany specimens delivered to the laboratory. The information on the form must match the information on the specimen label. Additional information on the form can include method of collection or type of specimen, possible interfering medications, and the patient’s clinical information. The time the specimen is received in the laboratory should be recorded on the form. Specimen Rejection Improperly labeled and collected specimens should be rejected by the laboratory, and appropriate personnel should be notified to collect a new specimen. Unacceptable situations include: 1. Specimens in unlabeled containers 2. Nonmatching labels and requisition forms 3. Specimens contaminated with feces or toilet paper 4. Containers with contaminated exteriors 5. Specimens of insufficient quantity 6. Specimens that have been improperly transported Laboratories should have a written policy detailing their conditions for specimen rejection. TECHNICAL TIP Never discard a specimen before checking with a supervisor. Table 2–2 The fact that a urine specimen is so readily available and easily collected often leads to laxity in the treatment of the specimen after its collection. Changes in urine composition take place not only in vivo but also in vitro, thus requiring correct handling procedures. Specimen Integrity Following collection, specimens should be delivered to the laboratory promptly and tested within 2 hours. A specimen that cannot be delivered and tested within 2 hours should be refrigerated or have an appropriate chemical preservative added. Table 2–2 describes the 11 most significant changes that may occur in a specimen allowed to remain unpreserved at room temperature for longer than 2 hours. Notice that most of the changes are related to the presence and growth of bacteria. These variations are discussed again under the individual test procedures. At this point it is important to realize that improper preservation can seriously affect the results of a routine urinalysis. Specimen Preservation The most routinely used method of preservation is refrigeration at 2°C to 8°C, which decreases bacterial growth and metabolism. If the urine is to be cultured, it should be refrigerated during transit and kept refrigerated until cultured up to 24 hours.2 The specimen must return to room temperature before chemical testing by reagent strips. When a specimen must be transported over a long distance and refrigeration is impossible, chemical preservatives may be added. Commercially prepared transport tubes are available. The ideal preservative should be bactericidal, inhibit urease, and preserve formed elements in the sediment. At the Changes in Unpreserved Urine Analyte Change Cause Color Clarity Odor pH Modified/darkened Decreased Increased Increased Glucose Ketones Bilirubin Urobilinogen Nitrite Red and white blood cells and casts Bacteria Trichomonas Decreased Decreased Decreased Decreased Increased Decreased Oxidation or reduction of metabolites Bacterial growth and precipitation of amorphous material Bacterial multiplication causing breakdown of urea to ammonia Breakdown of urea to ammonia by urease-producing bacteria/loss of CO2 Glycolysis and bacterial use Volatilization and bacterial metabolism Exposure to light/photo oxidation to biliverdin Oxidation to urobilin Multiplication of nitrate-reducing bacteria Disintegration in dilute alkaline urine Increased Decreased Multiplication Loss of motility, death 3920_Ch02_027-038 23/01/14 10:08 AM Page 32 32 Part One | Background TECHNICAL TIP Specimens must be returned to room temperature before chemical testing by reagent strips because the enzyme reactions on the strips perform best at room temperature. same time, the preservative should not interfere with chemical tests. Unfortunately, as can be seen in Table 2–3, the ideal preservative does not exist; therefore, a preservative that best suits the needs of the required analysis should be chosen. TECHNICAL TIP When preserving samples that will be transported to another laboratory, be sure to check with that laboratory concerning the appropriate preservative. Table 2–3 Types of Specimens To obtain a specimen that is representative of a patient’s metabolic state, regulation of certain aspects of specimen collection is often necessary. These special conditions may include time, length, and method of collection and the patient’s dietary and medicinal intake. It is important to instruct patients when they must follow special collection procedures. Frequently encountered specimens are listed in Table 2–4. Random Specimen This is the most commonly received specimen because of its ease of collection and convenience for the patient. The random specimen may be collected at any time, but the actual time of voiding should be recorded on the container.2 The random specimen is useful for routine screening tests to Urine Preservatives Preservatives Advantages Disadvantages Additional Information Refrigeration Does not interfere with chemical tests Prevents bacterial growth and metabolism Precipitates amorphous phosphates and urates Interferes with drug and hormone analyses Formalin (formaldehyde) Excellent sediment preservative Sodium fluoride Is a good preservative for drug analyses Commercial preservative tablets Yellow UA Plus tube Convenient when refrigeration not possible Have controlled concentration to minimize interference Contains collection cup, transfer straw, culture and sensitivity (C&S) preservative tube, or UA tube Sample stable at room temperature (RT) for 48 hours; prevents bacterial growth and metabolism Use on automated instruments Acts as a reducing agent, interfering with chemical tests for glucose, blood, leukocyte esterase, and copper reduction Inhibits reagent strip tests for glucose, blood, and leukocytes Check tablet composition to determine possible effects on desired tests Prevents bacterial growth for 24 hours2 Keeps pH at about 6.0 Can be used for urine culture transport Rinse specimen container with formalin to preserve cells and casts Cherry red/yellow Preservative Plus tube Stable for 72 hours at RT; instrument-compatible Boric acid Urine Collection Kits4 (Becton, Dickinson, Rutherford, NJ) Light gray and gray C&S tube Do not use if urine is below minimum fill line Must refrigerate within 2 hours Must be filled to minimum fill line. Bilirubin and urobilinogen may be decreased if specimen is exposed to light and left at RT Preservative is boric acid, sodium borate and sodium formate. Keeps pH at about 6.0 Round or conical bottom, no preservative Preservative is sodium propionate, ethyl paraben, and chlorhexidine; round or conical bottoms 3920_Ch02_027-038 23/01/14 10:08 AM Page 33 Chapter 2 | Introduction to Urinalysis 33 Table 2–4 Types of Urine Specimens Type of Specimen Purpose Random First morning Routine screening Routine screening Pregnancy tests Orthostatic protein Quantitative chemical tests Bacterial culture Routine screening Bacterial culture Bladder urine for bacterial culture Cytology Prostatic infection 24-hour (or timed) Catheterized Midstream clean-catch Suprapubic aspiration Three-glass collection detect obvious abnormalities. However, it may also show erroneous results resulting from dietary intake or physical activity just before collection. The patient will then be requested to collect an additional specimen under more controlled conditions. First Morning Specimen Although it may require the patient to make an additional trip to the laboratory, this is the ideal screening specimen. It is also essential for preventing false-negative pregnancy tests and for evaluating orthostatic proteinuria. The first morning specimen is a concentrated specimen, thereby assuring detection of chemicals and formed elements that may not be present in a dilute random specimen. The patient should be instructed to collect the specimen immediately on arising and to deliver it to the laboratory within 2 hours or keep it refrigerated. HISTORICAL NOTE Glucose Tolerance Specimens Glucose tolerance specimens are sometimes collected to correspond with the blood samples drawn during a glucose tolerance test (GTT). The number of specimens varies with the length of the test. GTTs may include fasting, half-hour, 1-hour, 2-hour, and 3-hour specimens, and possibly 4-hour, 5-hour, and 6-hour specimens. The urine is tested for glucose and ketones, and the results are reported along with the blood test results as an aid to interpreting the patient’s ability to metabolize a measured amount of glucose and are correlated with the renal threshold for glucose. Collection of these specimens is an institutional option.5 24-Hour (or Timed) Specimen Measuring the exact amount of a urine chemical is often necessary instead of just reporting its presence or absence. A carefully timed specimen must be used to produce accurate quantitative results. Many solutes exhibit diurnal variations such as catecholamines, 17-hydroxysteroids, and electrolytes in which the lowest concentration is in the early morning and the highest concentration occurs in the afternoon.2 When the concentration of the substance to be measured changes with diurnal variations and with daily activities such as exercise, meals, and body metabolism, 24-hour collection is required. If the concentration of a particular substance remains constant, the specimen may be collected over a shorter period. Care must be taken, however, to keep the patient adequately hydrated during short collection periods. Patients must be instructed on the procedure for collecting a timed specimen. To obtain an accurate timed specimen, the patient must begin and end the collection period with an empty bladder. The concentration of a substance in a particular period must be calculated from the urine volume produced during that time. On its arrival in the laboratory, a 24-hour specimen must be thoroughly mixed and the volume accurately measured and recorded. If only an aliquot is needed for testing, the amount saved must be adequate to permit repeat or additional testing. If a specimen is collected in two containers, the contents of the containers should be combined and thoroughly mixed before aliquoting. Consideration also must be given to the preservation of specimens collected over extended periods. All specimens should be refrigerated or kept on ice during the collection period and may also require addition of a chemical PROCEDURE 2-1 Sample 24-Hour (Timed) Specimen Collection Procedure Provide the patient with written instructions, and explain the collection procedure. Provide the patient with the proper collection container and preservative. Day 1: 7 a.m.: patient voids and discards specimen; collects all urine for the next 24 hours. Day 2: 7 a.m.: patient voids and adds this urine to previously collected urine. On arrival at laboratory, the entire 24-hour specimen is thoroughly mixed, and the volume is measured and recorded. TECHNICAL TIP Addition of urine formed before the start of the collection period will falsely elevate results and failure to include urine produced at the end of the collection period will falsely decrease results. 3920_Ch02_027-038 23/01/14 10:08 AM Page 34 34 Part One | Background preservative. The preservative chosen must be nontoxic to the patient and should not interfere with the tests to be performed. Appropriate collection information is included with test procedures and should be read before issuing a container and instructions to the patient. Catheterized Specimen This specimen is collected under sterile conditions by passing a hollow tube (catheter) through the urethra into the bladder. The most commonly requested test on a catheterized specimen is a bacterial culture. Midstream Clean-Catch Specimen As an alternative to the catheterized specimen, the midstream clean-catch specimen provides a safer, less traumatic method for obtaining urine for bacterial culture and routine urinalysis. It provides a specimen that is less contaminated by epithelial cells and bacteria and, therefore, is more representative of the actual urine than the routinely voided specimen. Patients must be provided with appropriate cleansing materials, a sterile container, and instructions for cleansing and voiding. Strong bacterial agents, such as hexachlorophene or povidone-iodine, should not be used as cleansing agents. Mild antiseptic towelettes are recommended. Some urine collection transfer kits contain Castile Soap Towelettes. PROCEDURE 2-2 Clean-Catch Specimen Collection: Female Cleansing Procedure2 1. Wash hands. 2. Remove the lid from the container without touching the inside of the container or lid. 3. Separate the skin folds (labia). 4. Cleanse from front to back on either side of the urinary opening with an antiseptic towelette, using a clean one for each side. 5. Hold the skin folds apart and begin to void into the toilet. 6. Bring the urine container into the stream of urine and collect an adequate amount of urine. Do not touch the inside of the container or allow the container to touch the genital area. 7. Finish voiding into the toilet. 8. Cover the specimen with the lid. Touch only the outside of the lid and container. 9. Label the container with the name and time of collection and place in the specified area or follow institutional policy. Suprapubic Aspiration Occasionally urine may be collected by external introduction of a needle through the abdomen into the bladder. Because the bladder is sterile under normal conditions, suprapubic aspiration provides a sample for bacterial culture that is completely free of extraneous contamination. The specimen can also be used for cytologic examination. Prostatitis Specimen Several methods are available to detect the presence of prostatitis. Three-Glass Collection Prior to collection the area is cleansed using the male midstream clean-catch procedure. Then instead of discarding the first urine passed, it is collected in a sterile container. Next, the midstream portion is collected in another sterile container. The prostate is then massaged so that prostate fluid will be passed with the remaining urine into a third sterile container. Quantitative cultures are performed on all specimens, and the first TECHNICAL TIP When both a routine urinalysis and a culture are requested on a catheterized or midstream collection, the culture should be performed first to prevent contamination of the specimen. A collection transfer kit can also be used. PROCEDURE 2-3 Clean-Catch Specimen Collection: Male Cleansing Procedure2 1. Wash hands. 2. Remove the lid from the sterile container without touching the inside of the container or lid. 3. Cleanse the tip of the penis with antiseptic towelette and let dry. Retract the foreskin if uncircumcised. 4. Void into the toilet. Hold back foreskin if necessary. 5. Bring the sterile urine container into the stream of urine and collect an adequate amount of urine. Do not touch the inside of the container or allow the container to touch the genital area. 6. Finish voiding into the toilet. 7. Cover the specimen with the lid. Touch only the outside of the lid and container. 8. Label the container with the name and time of collection and place in the specified area or follow institutional policy. 3920_Ch02_027-038 23/01/14 10:08 AM Page 35 Chapter 2 | Introduction to Urinalysis 35 and third specimens are examined microscopically. In prostatic infection, the third specimen will have a white blood cell/ high-power field count and a bacterial count 10 times that of the first specimen. Macrophages containing lipids may also be present. The second specimen is used as a control for bladder and kidney infection. If it is positive, the results from the third specimen are invalid because infected urine has contaminated the specimen.6 Pre- and Post-Massage Test In the pre- and post-massage test (PPMT), a clean-catch midstream urine specimen is collected. A second urine sample is collected after the prostate is massaged. A positive result is significant bacteriuria in the post-massage specimen of greater than 10 times the premassage count.7 Pediatric Specimens Collection of pediatric specimens can present a challenge. Soft, clear plastic bags with hypoallergenic skin adhesive to attach to the genital area of both boys and girls are available for collecting routine specimens. Sterile specimens may be obtained by catheterization or by suprapubic aspiration. Care must be taken not to touch the inside of the bag when applying it. For routine specimen analysis ensure the area is free of contamination. Attach the bag firmly over the genital area avoiding the anus. When enough specimen has been collected, remove the bag and label it or pour the specimen into container and label the container following institutional policy. For microbiology specimens clean the area with soap and water and sterilely dry the area, removing any residual soap residue. Firmly apply a sterile bag. Sterilely transfer collected specimen into a sterile container and label the container.2 HISTORICAL NOTE Stamey-Mears Test for Prostatitis The four-glass method consists of bacterial cultures of the initial voided urine (VB1), midstream urine (VB2), expressed prostatic secretions (EPS), and a post-prostatic massage urine specimen (VB3). Urethral infection or inflammation is tested for by the VB1, and the VB2 tests for urinary bladder infection. The prostatic secretions are cultured and examined for white blood cells. Having more than 10 to 20 white blood cells per high-power field is considered abnormal.7 TECHNICAL TIP Check the applied bags approximately every 15 minutes until the needed amount of sample has been collected. Drug Specimen Collection Urine specimen collection is the most vulnerable part of a drug-testing program. Correct collection procedures and documentation are necessary to ensure that the results are those of the specific individual submitting the specimen. The chain of custody (COC) is the process that provides this documentation of proper sample identification from the time of collection to the receipt of laboratory results. The COC is a standardized form that must document and accompany every step of drug testing, from collector to courier to laboratory to medical review officer to employer. For urine specimens to withstand legal scrutiny, it is necessary to prove that no tampering of the specimen occurred, such as substitution, adulteration, or dilution. All personnel handling the specimen must be noted. The specimen must be handled securely, with a guarantee that no unauthorized access to the specimen was possible. Proper identification of the individual whose information is indicated on the label is required. Either photo identification or positive identification by an employer representative with photo ID is acceptable. Urine specimen collections may be “witnessed” or “unwitnessed.” The decision to obtain a witnessed collection is indicated when it is suspected that the donor may alter or substitute the specimen or it is the policy of the client ordering the test. If a witnessed specimen collection is ordered, a same-gender collector will observe the collection of 30 to 45 mL of urine. Witnessed and unwitnessed collections should be immediately handed to the collector. The urine temperature must be taken within 4 minutes from the time of collection to confirm the specimen has not been adulterated. The temperature should read within the range of 32.5°C to 37.7°C. If the specimen temperature is not within range, the temperature should be recorded and the supervisor or employer contacted immediately. Urine temperatures outside of the recommended range may indicate specimen contamination. Recollection of a second specimen as soon as possible will be necessary. The urine color is also inspected to identify any signs of contaminants. The specimen is labeled, packaged, and transported following laboratory-specific instructions. 3920_Ch02_027-038 23/01/14 10:08 AM Page 36 36 Part One | Background PROCEDURE 2-4 Urine Drug Specimen Collection Procedure 1. The collector washes hands and wears gloves. 2. The collector adds bluing agent (dye) to the toilet water reservoir to prevent an adulterated specimen. 3. The collector eliminates any source of water other than toilet by taping the toilet lid and faucet handles. 4. The donor provides photo identification or positive identification from employer representative. 5. The collector completes step 1 of the chain-of-custody (COC) form and has the donor sign the form. 6. The donor leaves his or her coat, briefcase, and/or purse outside the collection area to avoid the possibility of concealed substances contaminating the urine. 7. The donor washes his or her hands and receives a specimen cup. 8. The collector remains in the restroom but outside the stall, listening for unauthorized water use, unless a witnessed collection is requested. 9. The donor hands specimen cup to the collector. Transfer is documented. 10. The collector checks the urine for abnormal color and for the required amount (30 to 45 mL). 11. The collector checks that the temperature strip on the specimen cup reads 32.5°C to 37.7°C. The collector Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Herman, JR: Urology: A View Through the Retrospectroscope. Harper & Row, Hagerstown, MD, 1973. 2. Clinical and Laboratory Standards Institute (formerly NCCLS), Approved Guideline GP16-A3: Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline—ed 3, CLSI, Wayne, PA, 2009. 12. 13. 14. 15. 16. 17. 18. 19. 20. records the in-range temperature on the COC form (COC step 2). If the specimen temperature is out of range or the specimen is suspected of having been diluted or adulterated, a new specimen must be collected and a supervisor notified. The specimen must remain in the sight of the donor and collector at all times. With the donor watching, the collector peels off the specimen identification strips from the COC form (COC step 3) and puts them on the capped bottle, covering both sides of the cap. The donor initials the specimen bottle seals. The date and time are written on the seals. The donor completes step 4 on the COC form. The collector completes step 5 on the COC form. Each time the specimen is handled, transferred, or placed in storage, every individual must be identified and the date and purpose of the change recorded. The collector follows laboratory-specific instructions for packaging the specimen bottles and laboratory copies of the COC form. The collector distributes the COC copies to appropriate personnel. 3. Torora, GJ, and Anagnostakos, NP: Principles of Anatomy and Physiology, ed 6, Harper & Row, New York, 1990, p. 51. 4. Becton, Dickinson and Company: BD Vacutainer Urine Products for collection, storage, and transport of urine specimens. Product Circular, 2011. 5. Baer, DM: Glucose tolerance test: Tips from the clinical experts. Medical Laboratory Observer, Sept. 2003. 6. Rous, SN: The Prostate Book. Consumers Union, Mt. Vernon, NY, 1988. 7. Stevermer, JJ, and Easley, SK: Treatment of prostatitis. Am Fam Physician 61(10), 2000. Study Questions 1. The average daily output of urine is: A. 200 mL B. 500 mL C. 1200 mL D. 2500 mL 2. An unidentified fluid is received in the laboratory with a request to determine whether the fluid is urine or another body fluid. Using routine laboratory tests, what tests would determine that the fluid is most probably urine? A. Glucose and ketones B. Urea and creatinine C. Uric acid and amino acids D. Protein and amino acids 3920_Ch02_027-038 23/01/14 10:08 AM Page 37 Chapter 2 | Introduction to Urinalysis 37 3. The primary inorganic substance found in urine is: A. Sodium B. Phosphate C. Chloride D. Calcium 4. A patient presenting with polyuria, nocturia, polydipsia, and a low urine specific gravity is exhibiting symptoms of: A. Diabetes insipidus B. Diabetes mellitus C. Urinary tract infection D. Uremia 5. A patient with oliguria might progress to having: A. Nocturia B. Polyuria C. Polydipsia D. Anuria 6. All of the following are characteristics of recommended urine containers except: A. A flat bottom B. A capacity of 50 mL C. A snap-on lid D. Are disposable 7. Labels for urine containers are: A. Attached to the container B. Attached to the lid C. Placed on the container prior to collection D. Not detachable 8. A urine specimen may be rejected by the laboratory for all of the following reasons except the fact that the: A. Requisition states the specimen is catheterized B. Specimen contains toilet paper C. Label and requisition do not match D. Outside of the container has fecal material contamination 9. A cloudy specimen received in the laboratory may have been preserved using: A. Boric acid B. Chloroform C. Refrigeration D. Formalin 10. For general screening the most frequently collected specimen is a: A. Random one B. First morning C. Midstream clean-catch D. Timed 11. The primary advantage of a first morning specimen over a random specimen is that it: A. Is less contaminated B. Is more concentrated C. Is less concentrated D. Has a higher volume 12. If a routine urinalysis and a culture are requested on a catheterized specimen, then: A. Two separate containers must be collected B. The routine urinalysis is performed first C. The patient must be recatheterized D. The culture is performed first 13. If a patient fails to discard the first specimen when collecting a timed specimen the: A. Specimen must be recollected B. Results will be falsely elevated C. Results will be falsely decreased D. Both A and B 14. The primary cause of unsatisfactory results in an unpreserved routine specimen not tested for 8 hours is: A. Bacterial growth B. Glycolysis C. Decreased pH D. Chemical oxidation 15. Prolonged exposure of a preserved urine specimen to light will cause: A. Decreased glucose B. Increased cells and casts C. Decreased bilirubin D. Increased bacteria 16. Which of the following would be least affected in a specimen that has remained unpreserved at room temperature for more than 2 hours? A. Urobilinogen B. Ketones C. Protein D. Nitrite 17. Bacterial growth in an unpreserved specimen will: A. Decrease clarity B. Increase bilirubin C. Decrease pH D. Increase glucose 18. The most sterile specimen collected is a: A. Catheterized B. Midstream clean-catch C. Three-glass D. Suprapubic aspiration 3920_Ch02_027-038 23/01/14 10:08 AM Page 38 38 Part One | Background 19. Which of the following would not be given to a patient prior to the collection of a midstream clean-catch specimen? A. Sterile container B. Iodine cleanser C. Antiseptic towelette D. Instructions 20. Urine specimen collection for drug testing requires the collector to do all of the following except: A. Inspect the specimen color B. Perform reagent strip testing C. Read the specimen temperature D. Fill out a chain-of-custody form Case Studies and Clinical Situations 1. A patient brings a first morning specimen to the laboratory at 1:00 p.m. a. How could this affect the urinalysis results? b. What could the patient say that would make the specimen satisfactory for testing? 2. A patient collecting a midstream clean-catch specimen voids immediately into the container. a. How could this affect the clarity of the specimen? b. How could this affect the microscopic examination? 3. A patient brings a 24-hour timed specimen to the laboratory and reports that he or she forgot to collect a specimen voided during the night. a. How will this affect the results of a quantitative test for creatinine? b. What should the patient be told to do? 4. You receive a urine preservative tube for culture containing a volume of specimen that is considerably below the minimum fill line. a. Could this affect the culture? b. Why? 5. A worker suspects that he or she will be requested to collect an unwitnessed urine specimen for drug analysis. He or she carries a substitute specimen in his or her pocket for 2 days before being told to collect the specimen. Shortly after the worker delivers the specimen to the collector, he or she is instructed to collect another specimen. a. What test was performed on the specimen to determine possible specimen manipulation? b. How was the specimen in this situation affected? c. If a specimen for drug analysis tests positive, state a possible defense related to specimen collection and handling that an attorney might employ. d. How can this defense be avoided? 3920_Ch03_039-057 23/01/14 10:10 AM Page 39 CHAPTER 3 Renal Function LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 3-1 Identify the components of the nephron, kidney, and excretory system. 3-10 Given hypothetical laboratory data, calculate a creatinine clearance and determine whether the result is normal. 3-2 Trace the flow of blood through the nephron and state the physiologic functions that occur. 3-11 Discuss the clinical significance of the glomerular filtration rate tests. 3-3 Describe the process of glomerular ultrafiltration. 3-12 Describe and contrast the MDRD, cystatin C, and beta2-microglobulin tests for performing estimated glomerular filtration rates (eGFR). 3-4 Discuss the functions and regulation of the reninangiotensin-aldosterone system. 3-5 Differentiate between active and passive transport in relation to renal concentration. 3-6 Explain the function of antidiuretic hormone in the concentration of urine. 3-7 Describe the role of tubular secretion in maintaining acid–base balance. 3-8 Identify the laboratory procedures used to evaluate glomerular filtration, tubular reabsorption and secretion, and renal blood flow. 3-9 Describe the creatinine clearance test. 3-13 Define osmolarity and discuss its relationship to urine concentration. 3-14 Describe the basic principles of freezing point osmometers. 3-15 Given hypothetic laboratory data, calculate a freewater clearance and interpret the result. 3-16 Given hypothetic laboratory data, calculate a PAH clearance and relate this result to renal blood flow. 3-17 Describe the relationship of urinary ammonia and titratable acidity to the production of an acidic urine. KEY TERMS Active transport Glomerular filtration barrier Renal plasma flow Afferent arteriole Glomerular filtration rate (GFR) Renal threshold Aldosterone Glomerulus Renin Antidiuretic hormone (ADH) Juxtaglomerular apparatus Beta2-microglobulin Loops of Henle Renin-angiotensin-aldosterone system (RAAS) Collecting duct Maximal reabsorptive capacity (Tm) Shield of negativity Countercurrent mechanism Nephron Titratable acidity Creatinine Osmolality Tubular reabsorption Cystatin C Passive transport Tubular secretion Distal convoluted tubule Peritubular capillaries Vasa recta Efferent arteriole Podocytes Free water clearance Proximal convoluted tubule 3920_Ch03_039-057 23/01/14 10:10 AM Page 40 40 Part One | Background This chapter reviews nephron anatomy and physiology and discusses their relationship to urinalysis and renal function testing. A section on laboratory assessment of renal function is included. Renal Physiology Each kidney contains approximately 1 to 1.5 million functional units called nephrons. As shown in Figure 3–1, the human kidney contains two types of nephrons. Cortical nephrons, which make up approximately 85% of nephrons, are situated primarily in the cortex of the kidney. They are responsible primarily for removal of waste products and reabsorption of nutrients. Juxtamedullary nephrons have longer loops of Henle that extend deep into the medulla of the kidney. Their primary function is concentration of the urine. The ability of the kidneys to clear waste products selectively from the blood and simultaneously to maintain the body’s essential water and electrolyte balances is controlled in the nephron by the following renal functions: renal blood flow, glomerular filtration, tubular reabsorption, and tubular secretion. The physiology, laboratory testing, and associated pathology of these four functions are discussed in this chapter. Renal Blood Flow The renal artery supplies blood to the kidney. The human kidneys receive approximately 25% of the blood pumped through the heart at all times. Blood enters the capillaries of the Glomerulus Renal cortex Cortical nephron Renal tubule Papilla of pyramid Renal artery Renal medulla Loop of Henle Juxtamedullary nephron Collecting duct Calyx Renal vein Cortex Renal pelvis Ureter Urinary bladder Urethra Figure 3–1 The relationship of the nephron to the kidney and excretory system. (From Scanlon, VC, and Sanders, T: Essentials of Anatomy and Physiology, ed 3. FA Davis, Philadelphia, PA, 1999, p 405, with permission.) 3920_Ch03_039-057 23/01/14 10:10 AM Page 41 Chapter 3 | Renal Function nephron through the afferent arteriole. It then flows through the glomerulus and into the efferent arteriole. The varying sizes of these arterioles help to create the hydrostatic pressure differential important for glomerular filtration and to maintain consistency of glomerular capillary pressure and renal blood flow within the glomerulus. Notice the smaller size of the efferent arteriole in Figure 3–2. This increases the glomerular capillary pressure. Before returning to the renal vein, blood from the efferent arteriole enters the peritubular capillaries and the vasa recta and flows slowly through the cortex and medulla of the kidney close to the tubules. The peritubular capillaries surround the proximal and distal convoluted tubules, providing for the immediate reabsorption of essential substances from the fluid in the proximal convoluted tubule and final adjustment of the urinary composition in the distal convoluted tubule. The vasa recta are located adjacent to the ascending and descending loops of Henle in juxtamedullary nephrons. In this area, the major exchanges of water and salts take place between the blood and the medullary interstitium. This exchange maintains the osmotic gradient (salt concentration) in the medulla, which is necessary for renal concentration. Based on an average body size of 1.73 m2 of surface, the total renal blood flow is approximately 1200 mL/min, and the total renal plasma flow ranges from 600 to 700 mL/min. Normal values for renal blood flow and renal function tests depend on body size. When dealing with sizes that vary greatly from the average 1.73 m2 of body surface, a correction must 41 be calculated to determine whether the observed measurements represent normal function. This calculation is covered in the discussion on tests for glomerular filtration rate (GFR) later in this chapter. Variations in normal values have been published for different age groups and should be considered when evaluating renal function studies. Glomerular Filtration The glomerulus consists of a coil of approximately eight capillary lobes, the walls of which are referred to as the glomerular filtration barrier. It is located within Bowman’s capsule, which forms the beginning of the renal tubule. Although the glomerulus serves as a nonselective filter of plasma substances with molecular weights less than 70,000, several factors influence the actual filtration process. These include the cellular structure of the capillary walls and Bowman’s capsule, hydrostatic pressure and oncotic pressure, and the feedback mechanisms of the renin-angiotensin-aldosterone system (RAAS). Cellular Structure of the Glomerulus Plasma filtrate must pass through three glomerular filtration barrier cellular layers: the capillary wall membrane, the basement membrane (basal lamina), and the visceral epithelium of Bowman’s capsule. The endothelial cells of the capillary wall differ from those in other capillaries by containing pores and are referred to as fenestrated. The pores increase capillary permeability but do not allow the passage of large molecules and Afferent arteriole Juxtaglomerular apparatus Efferent arteriole Bowman’s capsule Glomerulus Distal convoluted tubule Proximal convoluted tubule Peritubular capillaries Thick descending loop of Henle Vasa recta Thin descending loop of Henle Figure 3–2 The nephron and its component parts. Cortex Collecting duct Vasa recta Medulla Thick ascending loop of Henle Thin ascending loop of Henle 3920_Ch03_039-057 23/01/14 10:10 AM Page 42 42 Part One | Background blood cells. Further restriction of large molecules occurs as the filtrate passes through the basement membrane and the thin membranes covering the filtration slits formed by the intertwining foot processes of the podocytes of the inner layer of Bowman’s capsule (see Fig. 3–3A). In addition to the structure of the glomerular filtration barrier that prohibits the filtration of large molecules, the barrier contains a shield of negativity that repels molecules with a positive charge even though they are small enough to pass through the three layers of the barrier. The shield is very important because albumin (the primary protein associated with renal disease) has a positive charge and would easily pass through the barrier (see Fig. 3–3B). Glomerular Pressure As mentioned previously, the presence of hydrostatic pressure resulting from the smaller size of the efferent arteriole and the glomerular capillaries enhances filtration. This pressure is necessary to overcome the opposition of pressures from the fluid within Bowman’s capsule and the oncotic pressure of unfiltered plasma proteins in the glomerular capillaries. By increasing or decreasing the size of the afferent and efferent arterioles, an autoregulatory mechanism within the juxtaglomerular apparatus maintains the glomerular blood pressure at a relatively constant rate regardless of fluctuations in systemic blood pressure. Dilation of the afferent arterioles and constriction of the TECHNICAL TIP If it were not for the shield of negativity, all routine urines would have positive reagent strip readings for protein/albumin. efferent arterioles when blood pressure drops prevents a marked decrease in blood flowing through the kidney, thus preventing an increase in the blood level of toxic waste products. Likewise, an increase in blood pressure results in constriction of the afferent arterioles to prevent overfiltration or damage to the glomerulus. Renin-Angiotensin-Aldosterone System The renin-angiotensin-aldosterone system regulates the flow of blood to and within the glomerulus. The system responds to changes in blood pressure and plasma sodium content that are monitored by the juxtaglomerular apparatus, which consists of the juxtaglomerular cells in the afferent arteriole and the macula densa of the distal convoluted tubule (Fig. 3–4). Low plasma sodium content decreases water retention within the circulatory system, resulting in a decreased overall blood volume and subsequent decrease in blood pressure. When the macula densa senses such changes, a cascade of reactions within the RAAS occurs (Fig. 3–5). Renin, an enzyme produced by the juxtaglomerular cells, is secreted and reacts with the blood-borne substrate angiotensinogen to produce the inert hormone angiotensin I. As angiotensin I passes through the alveoli of the lungs, angiotensin-converting enzyme (ACE) changes it to the active form angiotensin II. Angiotensin II corrects renal blood flow in the following ways: causing vasodilation of the afferent arterioles and constriction of the efferent arterioles, stimulating reabsorption of sodium and water in the proximal convoluted tubules, and triggering the release of the sodium-retaining hormone aldosterone by the adrenal cortex and antidiuretic hormone by the hypothalamus (Table 3–1). As systemic blood pressure and plasma sodium content increase, the secretion of renin decreases. Therefore, the actions Afferent arteriole Efferent arteriole Glomerular basement membrane Hydrostatic pressure Oncotic pressure (unfiltered plasma proteins) Protein Glomerular basement membrane Foot processes of podocyte B Fenestrated epithelium Slit diaphragm Podocyte foot process Podocyte Bowman’s space Proximal convoluted tubule Blood A C Glomerular basement membrane Fenestrated endothelium Figure 3–3 Factors affecting glomerular filtration in the renal corpuscle (A). Inset B, glomerular filtration barrier. Inset C, the shield of negativity. 3920_Ch03_039-057 23/01/14 10:10 AM Page 43 Chapter 3 | Renal Function Afferent arteriole Distal tubule Macula densa Table 3–1 Efferent arteriole Juxtaglomerular cells Glomerulus Figure 3–4 Close contact of the distal tubule with the afferent arteriole, macula densa, and the juxtaglomerular cells within the juxtaglomerular apparatus. Note the smaller size of the afferent arteriole indicating increased blood pressure. of angiotensin II produce a constant pressure within the nephron. As a result of the above glomerular mechanisms, every minute approximately two to three million glomeruli filter approximately 120 mL of water-containing low-molecular-weight substances. Because this filtration is nonselective, the only difference between the compositions of the filtrate and the plasma 43 Actions of the RAAS 1. Dilates the afferent arteriole and constricts the efferent arteriole 2. Stimulates sodium reabsorption in the proximal convoluted tubule 3. Triggers the adrenal cortex to release the sodiumretaining hormone aldosterone to cause sodium reabsorption and potassium excretion in the distal convoluted tubule and collecting duct 4. Triggers antidiuretic hormone release by the hypothalamus to stimulate water reabsorption in the collecting duct is the absence of plasma protein, any protein-bound substances, and cells. Analysis of the fluid as it leaves the glomerulus shows the filtrate to have a specific gravity of 1.010 and confirms that it is chemically an ultrafiltrate of plasma. This information provides a useful baseline for evaluating the renal mechanisms involved in converting the plasma ultrafiltrate into the final urinary product. Tubular Reabsorption The body cannot lose 120 mL of water-containing essential substances every minute. Therefore, when the plasma ultrafiltrate enters the proximal convoluted tubule, the nephrons, through cellular transport mechanisms, begin reabsorbing these essential substances and water (Table 3–2). Reabsorption Mechanisms The cellular mechanisms involved in tubular reabsorption are termed active transport and passive transport. For active transport to occur, the substance to be reabsorbed must combine with a carrier protein contained in the membranes of the Low blood pressure Low plasma sodium Renin secretion Angiotensinogen Angiotensin I Angiotensin converting enzymes Angiotensin II Vasoconstriction Figure 3–5 Algorithm of the reninangiotensin-aldosterone system. Proximal convoluted tubule Sodium reabsorption Aldosterone ADH Distal convoluted tubule Sodium reabsorption Collecting duct Water resorption 3920_Ch03_039-057 23/01/14 10:10 AM Page 44 44 Part One | Background Table 3–2 Active transport Passive transport Tubular Reabsorption Substance Location Glucose, amino acids, salts Chloride Sodium Proximal convoluted tubule Water Urea Sodium Ascending loop of Henle Proximal and distal convoluted tubules Proximal convoluted tubule Descending loop of Henle Collecting duct Proximal convoluted tubule Ascending loop of Henle Ascending loop of Henle renal tubular epithelial cells. The electrochemical energy created by this interaction transfers the substance across the cell membranes and back into the bloodstream. Active transport is responsible for the reabsorption of glucose, amino acids, and salts in the proximal convoluted tubule, chloride in the ascending loop of Henle, and sodium in the distal convoluted tubule. Passive transport is the movement of molecules across a membrane as a result of differences in their concentration or electrical potential on opposite sides of the membrane. These physical differences are called gradients. Passive reabsorption of water takes place in all parts of the nephron except the ascending loop of Henle, the walls of which are impermeable to water. Urea is passively reabsorbed in the proximal convoluted tubule and the ascending loop of Henle, and passive reabsorption of sodium accompanies the active transport of chloride in the ascending loop. Active transport, like passive transport, can be influenced by the concentration of the substance being transported. When the plasma concentration of a substance that is normally completely reabsorbed reaches an abnormally high level, the filtrate concentration exceeds the maximal reabsorptive capacity (Tm) of the tubules, and the substance begins appearing in the urine. The plasma concentration at which active transport stops is termed the renal threshold. For glucose, the plasma renal threshold is 160 to 180 mg/dL, and glucose appears in the urine when the plasma concentration reaches this level. Knowledge of the renal threshold and the plasma concentration can be used to distinguish between excess solute filtration and renal tubular damage. Active transport of more than two-thirds of the filtered sodium out of the proximal convoluted tubule is accompanied by the passive reabsorption of an equal amount of water. Therefore, as can be seen in Figure 3–6, the fluid leaving the proximal convoluted tubule still maintains the same concentration as the ultrafiltrate. TECHNICAL TIP Glucose appearing in the urine of a person with a normal blood glucose level is the result of tubular damage and not diabetes mellitus. A nonfasting patient with high glucose intake would not have a normal blood glucose. Tubular Concentration Renal concentration begins in the descending and ascending loops of Henle, where the filtrate is exposed to the high osmotic gradient of the renal medulla. Water is removed by osmosis in the descending loop of Henle, and sodium and chloride are reabsorbed in the ascending loop. Excessive reabsorption of water as the filtrate passes through the highly concentrated medulla is prevented by the water-impermeable walls of the ascending loop. This selective reabsorption process is called the countercurrent mechanism and serves to maintain the osmotic gradient of the medulla (see Fig. 3–6). The sodium and chloride leaving the filtrate in the ascending loop prevent dilution of the medullary interstitium by the water reabsorbed from the descending loop. Maintenance of this osmotic gradient is essential for the final concentration of the filtrate when it reaches the collecting duct. In Figure 3–6, the actual concentration of the filtrate leaving the ascending loop is quite low owing to the reabsorption of salt and not water in that part of the tubule. Reabsorption of sodium continues in the distal convoluted tubule, but it is now under the control of the hormone aldosterone, which regulates reabsorption in response to the body’s need for sodium (see Fig. 3–5). Collecting Duct Concentration The final concentration of the filtrate through the reabsorption of water begins in the late distal convoluted tubule and continues in the collecting duct. Reabsorption depends on the osmotic gradient in the medulla and the hormone vasopressin (antidiuretic hormone [ADH]). One would expect that as the dilute filtrate in the collecting duct comes in contact with the higher osmotic concentration of the medullary interstitium, passive reabsorption of water would occur. However, the process is controlled by the presence or absence of ADH, which renders the walls of the distal convoluted tubule and collecting duct permeable or impermeable to water. A high level of ADH increases permeability, resulting in increased reabsorption of water, and a low-volume concentrated urine. Likewise, absence of ADH renders the walls impermeable to water, resulting in a large volume of dilute urine. Just as the production of aldosterone is controlled by the body’s sodium concentration, production of ADH is determined by the state of body hydration. Therefore, the chemical balance in the body is actually the final determinant of urine volume and concentration. The concept of ADH control can be summarized in the following manner: ↑Body Hydration = ↓ADH = ↑Urine Volume ↓Body Hydration = ↑ADH = ↓Urine Volume 3920_Ch03_039-057 23/01/14 10:10 AM Page 45 Chapter 3 | Renal Function Cortex 45 Blood (vein) 300 mOsm/L Efferent arteriole Proximal convoluted tubule 300 Glomerulus Na+Cl- H2O 300 H2O 100 Descending limb H2O 600 Na+ClThick ascending limb Na+ Na+Cl- H2O H2O Na+ H2O 900 H2O H2O Loop of Henle 1200 Medulla Countercurrent mechanism Reabsorption Secretion Variable reabsorption Figure 3–6 Renal concentration. Tubular Secretion In contrast to tubular reabsorption, in which substances are removed from the glomerular filtrate and returned to the blood, tubular secretion involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate (Fig. 3–7). Tubular secretion serves two major functions: eliminating waste products not filtered by the glomerulus and regulating the acid–base balance in the body through the secretion of hydrogen ions. Many foreign substances, such as medications, cannot be filtered by the glomerulus because they are bound to plasma proteins. When these protein-bound substances enter the peritubular capillaries, they develop a stronger affinity for the tubular cells and dissociate from their carrier proteins, which results in their transport into the filtrate by the tubular cells. The major site for removal of these nonfiltered substances is the proximal convoluted tubule. Efferent arteriole Afferent arteriole Bowman’s capsule Glomerular filtrate Reabsorption To blood Secretion Tubule To urine Figure 3–7 Summary of movement of substances in the nephron. 3920_Ch03_039-057 23/01/14 10:10 AM Page 46 46 Part One | Background Acid–Base Balance Tubular lumen To maintain the normal blood pH of 7.4, the blood must buffer and eliminate the excess acid formed by dietary intake and body metabolism. The buffering capacity of the blood depends on bicarbonate (HCO3–) ions, which are readily filtered by the glomerulus and must be expediently returned to the blood to maintain the proper pH. As shown in Figure 3–8, the secretion of hydrogen ions (H+) by the renal tubular cells into the filtrate prevents the filtered bicarbonate from being excreted in the urine and causes the return of a bicarbonate ion to the plasma. This process provides for almost 100% reabsorption of filtered bicarbonate and occurs primarily in the proximal convoluted tubule. As a result of their small molecular size, hydrogen ions are readily filtered and reabsorbed. Therefore, the actual excretion of excess hydrogen ions also depends on tubular secretion. Figures 3–9 and 3–10 are diagrams of the two primary methods for hydrogen ion excretion in the urine. In Figure 3–9 the secreted hydrogen ion combines with a filtered phosphate ion instead of a bicarbonate ion and is excreted rather than reabsorbed. In the proximal convoluted tubule, ammonia is produced from the breakdown of the amino acid glutamine. The ammonia reacts with the H+ to form the ammonium ion Tubular lumen Renal tubular cell Peritubular plasma HCO3– (filtered) HCO3 + H+ H+ HCO3 – HCO3 – H2CO3 H2CO3 Carbonic anhydrase H2O + CO2 H2O + CO2 Renal tubular cell Peritubular plasma NH3 NH3 + H+ H+ HCO3 – HCO3 – H2CO3 NH4+ Carbonic anhydrase H2O + CO2 CO2 Final urine Figure 3–10 Excretion of secreted hydrogen ions combined with ammonia produced by the tubules. (NH4+) (see Fig. 3–10). The resulting ammonium ion is excreted in the urine. Should there be additional need for elimination of hydrogen ions, the distal convoluted tubule and the collecting duct are also able to produce ammonium ion. All three of these processes occur simultaneously at rates determined by the acid–base balance in the body. A disruption in these secretory functions can result in metabolic acidosis or renal tubular acidosis, the inability to produce an acid urine. Renal Function Tests This brief review of renal physiology shows that there are many metabolic functions and chemical interactions to be evaluated through laboratory tests of renal function. In Figure 3–11, the parts of the nephron are related to the laboratory tests used to assess their function. PAH CO2 Osmolarity GFR tests Final urine Figure 3–8 Reabsorption of filtered bicarbonate. Tubular lumen Renal tubular cell Ammonia Peritubular plasma HPO4– (filtered) HPO4– + H+ H+ HCO3– HCO3– H2CO3 H2PO4 Titratable acidity Free water clearance Carbonic anhydrase Osmolarity H2O + CO2 CO2 Final urine Figure 3–9 Excretion of secreted hydrogen ions combined with phosphate. Figure 3–11 The relationship of nephron areas to renal function tests. 3920_Ch03_039-057 23/01/14 10:10 AM Page 47 Chapter 3 | Renal Function Glomerular Filtration Tests The standard tests used to measure the filtering capacity of the glomeruli are termed clearance tests. As its name implies, a clearance test measures the rate at which the kidneys are able to remove (to clear) a filterable substance from the blood. To ensure that glomerular filtration is being measured accurately, the substance analyzed must be one that is neither reabsorbed nor secreted by the tubules. Other factors to consider in selecting a clearance test substance include the stability of the substance in urine during a possible 24-hour collection period, the plasma level consistency, the substance’s availability to the body, and the availability of tests to analyze the substance. Clearance Tests A variety of substances have been used to measure the GFR. Newer methods that eliminate many of the problems mentioned above have replaced some of these tests. They are summarized as Historical Notes. At present, creatinine, beta2-microglobulin, cystatin C, and possibly radioisotopes are the primary substances used in clearance tests. Each procedure has its advantages and disadvantages. A test that requires an infused substance is termed an exogenous procedure and is seldom the method of choice if a suitable test substance is already present in the body (endogenous procedure). HISTORICAL NOTE Urea Clearance The earliest glomerular filtration tests measured urea because of its presence in all urine specimens and the existence of routinely used methods of chemical analysis. Because approximately 40% of the filtered urea is reabsorbed, normal values were adjusted to reflect the reabsorption, and patients were hydrated to produce a urine flow of 2 mL/min to ensure that no more than 40% of the urea was reabsorbed. HISTORICAL NOTE Inulin Clearance Inulin, a polymer of fructose, is an extremely stable substance that is not reabsorbed or secreted by the tubules. It is not a normal body constituent, however, and must be infused by IV at a constant rate throughout the testing period. Therefore, although inulin was the original reference method for clearance tests, current methods are available that are endogenous and can provide accurate GFR results. 47 Creatinine Clearance Creatinine is a waste product of muscle metabolism that is produced enzymatically by creatine phosphokinase from creatine, which links with ATP to produce ADP and energy. Because creatinine is normally found at a relatively constant level in the blood, it provides the laboratory with an endogenous procedure for evaluating glomerular function. The use of creatinine has several disadvantages and careful consideration should be given to them. They are as follows: 1. Some creatinine is secreted by the tubules, and secretion increases as blood levels rise. 2. Chromogens present in human plasma react in the chemical analysis. Their presence, however, may help counteract the falsely elevated rates caused by tubular secretion. 3. Medications, including gentamicin, cephalosporins, and cimetidine (Tagamet), inhibit tubular secretion of creatinine, thus causing falsely low serum levels.1 4. Bacteria will break down urinary creatinine if specimens are kept at room temperature for extended periods.2 5. A diet heavy in meat consumed during collection of a 24-hour urine specimen will influence the results if the plasma specimen is drawn before the collection period because the increased intake of meat can raise the urine and plasma levels of creatinine during the 24-hour collection period. 6. Measurement of creatinine clearance is not a reliable indicator in patients suffering from muscle-wasting diseases or persons involved in heavy exercise or athletes supplementing with creatine. 7. Accurate results depend on the accurate completeness of a 24-hour collection. 8. It must be corrected for body surface area, unless normal is assumed, and must always be corrected for children. Newer methods that do not require the collection of timed (24-hour) urine specimens have been developed using just the serum creatinine, cystatin C, or beta2-microglobulin values. The results of these tests are reported as estimated glomerular filtration rate (eGFR). The traditional creatinine clearance procedure is included here because it is still being performed and its principles apply to other clearance procedures using urine. Procedure By far the greatest source of error in any clearance procedure using urine is the use of improperly timed urine specimens. The importance of using an accurately timed specimen (see Chapter 2) will become evident in the following discussion of the calculations involved in converting isolated laboratory measurements to the GFR. The GFR is reported in milliliters cleared per minute; therefore, determining the number of milliliters of plasma from which the clearance substance (creatinine) is completely removed during 1 minute is necessary. To calculate this information, one must know urine volume in 3920_Ch03_039-057 23/01/14 10:10 AM Page 48 48 Part One | Background mL/min (V), urine creatinine concentration in mg/dL (U), and plasma creatinine concentration in mg/dL (P). The urine volume is calculated by dividing the number of milliliters in the specimen by the number of minutes used to collect the specimen. EXAMPLE Calculate the urine volume (V) for a 2-hour specimen measuring 240 mL: 2 hours × 60 minutes = 120 minutes 240 mL/120 minutes = 2 mL/min V = 2 mL/min The plasma and urine concentrations are determined by chemical testing. The standard formula used to calculate the milliliters of plasma cleared per minute (C) is: C= UV 1.73 × P A with A being the actual body size in square meters of surface. The actual body size may be calculated as: log A = (0.425 × log weight) + (0.725 × log height) – 2.144 or it may be obtained from the nomogram shown in Figure 3–13. Estimated Glomerular Filtration Rates 1440 mL = 1 mL/min 60 minutes × 24 = 1440 minutes In the past years a variety of formulas for estimating glomerular filtration rates (eGFR) have been used and they continue to be revised. Because the formulas can be programmed into automated instruments, estimated clearances can be used for routinely screening patients as part of a metabolic profile and also to monitor patients already diagnosed with renal disease or at risk for renal disease. In addition, the formulas are valuable when medications that require adequate renal clearance need to be prescribed. The most frequently used formula is called the Modification of Diet in Renal Disease (MDRD) study. The formula has been modified several times to make it more accurate and standardized. At the present time the formula recommended by the National Kidney Disease Education Program (NKDEP) is called the MDRD-IDMS-traceable formula. A primary discrepancy in the previous formulas was found to be the methods used to measure serum creatinine. Current laboratory methods primarily use creatinine assays such as enzyme assays that do not have the same interference as the original Jaffe chemical method. These methods correspond more closely to the isotope dilution mass spectrophotometry (IDMS) reference method. The MDRD-IDMS traceable formula is: 120 mg/dL × 1 mL/min (V) = 120 mL/dL 1.0 mg/dL (P) GFR = 175 × serum creatinine –1154 × age –0.203 × 0.742 (if patient is female) × 1.202 (if patient is black) C = UV P This formula is derived as follows. The milliliters of plasma cleared per minute (C) times the mg/dL of plasma creatinine (P) must equal the mg/dL of urine creatinine (U) times the urine volume in mL/min (V), because all of the filtered creatinine will appear in the urine. Therefore: CP = UV and C = UV P EXAMPLE Using urine creatinine of 120 mg/dL (U), plasma creatinine of 1.0 mg/dL (P), and urine volume of 1440 mL obtained from a 24-hour specimen (V), calculate the GFR. V = the calculation when dealing with body sizes that deviate greatly from 1.73 m2 of surface, such as with children. To adjust a clearance for body size, the formula is: C = By analyzing this calculation and referring to Figure 3–12, at a 1 mg/dL concentration, each milliliter of plasma contains 0.01 mg creatinine. Therefore, to arrive at a urine concentration of 120 mg/dL (1.2 mg/mL), it is necessary to clear 120 mL of plasma. Although the filtrate volume is reduced, the amount of creatinine in the filtrate does not change because the creatinine is not reabsorbed. Knowing that in the average person (1.73 m2 body surface) the approximate amount of plasma filtrate produced per minute is 120 mL, it is not surprising that normal creatinine clearance values approach 120 mL/min (men, 107 to 139 mL/min; women, 87 to 107 mL/min). The normal reference range of plasma creatinine is 0.5 to 1.5 mg/dL. These reference values take into account variations in size and muscle mass. Values are considerably lower in older people, however, and an adjustment may also have to be made to Plasma (1 mg/dL = 0.01 mg/mL creatinine) Glomerulus Plasma filtrate (120 mL/min x 0.01 mg/mL = 1.2 mg) Reabsorption (119 mL H2O) Urine (1 mL/min) Creatinine (1.2 mg/mL or 120 mg/dL) Figure 3–12 Creatinine filtration and excretion. 3920_Ch03_039-057 23/01/14 10:10 AM Page 49 Chapter 3 | Renal Function 5' 10" 8" Height in feet 6" 4" 2" 4' 10" 8" 6" 4" 2" 150 145 140 135 130 125 120 115 110 105 1.05 180 170 180 160 150 140 120 110 140 .95 95 90 80 75 70 65 60 120 55 110 50 100 45 40 .90 95 .80 .75 80 35 70 30 .70 85 60 .65 8" 80 75 .60 Formula for MDRD calculation of GFR when the serum creatinine method is not standardized to IDMS. GFR = 173 × serum creatinine–1.154 × age–0.203 × 0.742 (if patient is female) × 1.212 (if patient is black) Cystatin C 85 130 90 Original MDRD Calculation 100 160 150 HISTORICAL NOTE 130 1.00 .85 90 6" 1.10 100 3' 10" 1.15 190 190 Weight in kilograms 2" 160 155 170 300 290 280 270 260 250 240 230 220 210 200 Weight in pounds 6" 4" 380 360 320 2.10 2.00 1.95 1.90 1.85 1.80 1.75 1.70 1.65 1.60 1.55 1.50 1.45 1.40 1.35 1.30 1.25 1.20 200 340 2.30 2.20 Surface area in square meters 6' 10" 8" 220 215 210 205 200 195 190 185 180 175 170 165 Height in centimeters 7' 10" 8" 6" 4" 2" 3.00 2.90 2.80 2.70 2.60 2.50 2.40 440 420 400 49 25 50 .55 20 .50 40 15 Figure 3–13 A nomogram for determining body surface area. (From Boothby, WM, and Sandiford, RB: Nomogram for determination of body surface area. N Engl J Med 185:227, 1921, with permission.) The formula is designed to essentially equal the results that compare to the reference body size of 1.73 m2. Because eGFRs are calculated for an average body size they are not accurate for pediatric patients. They have also been shown to be most accurate when results are lower than 60 mL/min.3 It is recommended that results be reported with numerical values below 60 mL/min and higher values reported as equal to or greater than 60 mL/min. The formula recommended for use when serum creatinine methods do not compare to the IDSM standard is provided as a Historical Note. Measurement of serum cystatin C has been shown to provide a good procedure for screening and monitoring GFR. Cystatin C is a small protein (molecular weight 13,359) produced at a constant rate by all nucleated cells. It is readily filtered by the glomerulus and reabsorbed and broken down by the renal tubular cells. Therefore, no cystatin C is secreted by the tubules, and the serum concentration can be directly related to the GFR. Immunoassay procedures are available for measuring cystatin C.4 Monitoring levels of cystatin C is recommended for pediatric patients, persons with diabetes, the elderly, and critically ill patients.5 An advantage of cystatin C is that it is independent of muscle mass. Recent studies also have shown that measuring both serum or plasma cystatin C and creatinine can provide even more accurate information on a patient’s GFR.6 Beta2-Microglobulin Beta2-microglobulin (molecular weight 11,800) dissociates from human leukocyte antigens at a constant rate and is rapidly removed from the plasma by glomerular filtration. Sensitive methods using enzyme immunoassay are available for the measurement of beta2-microglobulin. A rise in the plasma level of beta2-microglobulin has been shown to be a more sensitive indicator of a decrease in GFR than creatinine clearance. However, the test is not reliable in patients who have a history of immunologic disorders or malignancy.7 Radionucleotides Although they are exogenous procedures and more labor intensive and costly, injecting radionucleotides such as 125Iiothalamate provides a method for determining glomerular filtration through the plasma disappearance of the radioactive material and enables visualization of the filtration in one or both kidneys.8 This procedure can be valuable to measure the viability of a transplanted kidney. Clinical Significance When interpreting the results of a creatinine clearance test, the GFR is determined not only by the number of functioning nephrons but also by the functional capacity of these nephrons. In other words, even though half of the available nephrons may be nonfunctional, a change in the GFR will not occur if the remaining nephrons double their filtering capacity. This is 3920_Ch03_039-057 23/01/14 10:10 AM Page 50 50 Part One | Background evidenced by persons who lead normal lives with only one kidney. Therefore, although the GFR is a frequently requested laboratory procedure, its value does not lie in the detection of early renal disease. Instead, it is used to determine the extent of nephron damage in known cases of renal disease, to monitor the effectiveness of treatment designed to prevent further nephron damage, and to determine the feasibility of administering medications, which can build up to dangerous blood levels if the GFR is markedly reduced. the volume and specific gravity of day and night urine samples to evaluate concentrating ability. Neither test is used now because the information provided by specific gravity measurements is most useful as a screening procedure, and quantitative measurement of renal concentrating ability is best assessed through osmometry. Currently renal concentrating testing is performed after various periods of fluid deprivation, measuring urine and often serum osmolality. Controlled intake procedures can include after dinner overnight deprivation of fluid for 12 hours followed by collection of a urine sample. A urine osmolality reading of 800 mOsm or higher is normal and the test can be discontinued. If the urine test is abnormal, the fluid is restricted for another two hours and both urine and serum species are collected for osmolality testing. A urine to serum ratio (U:S ratio) of 3:1 or greater or a urine osmolality of 800 mOsm or greater indicates normal tubular reabsorption. If the test continues to be abnormal, additional testing is performed to determine whether the failure to concentrate the urine is caused by diabetes insipidus that occurs as the result of a problem with the production or the response of the kidney to ADH. The patient is injected with ADH and serum and urine specimens are collected in 2 and 4 hours. If at this time the test is normal, it indicates that the patient is not capable of producing ADH (neurogenic diabetes insipidus) and if the test is abnormal then the renal tubules are not responding to ADH (nephrogenic diabetes insipidus). See Figure 3–15. Tubular Reabsorption Tests Whereas measurement of the GFR is not a useful indication of early renal disease, the loss of tubular reabsorption capability is often the first function affected in renal disease. This is not surprising when one considers the complexity of the tubular reabsorption process. Tests to determine the ability of the tubules to reabsorb the essential salts and water that have been nonselectively filtered by the glomerulus are called concentration tests. As mentioned, the ultrafiltrate that enters the tubules has a specific gravity of 1.010; therefore, after reabsorption one would expect the final urine product to be more concentrated. However, as you perform routine urinalysis, you will see that many specimens do not have a specific gravity higher than 1.010, yet no renal disease is present. This is because urine concentration is largely determined by the body’s state of hydration, and the normal kidney will reabsorb only the amount of water necessary to preserve an adequate supply of body water. As can be seen in Figure 3–14, both specimens contain the same amount of solute; however, the urine density (specific gravity) of patient A will be higher. Therefore, control of fluid intake must be incorporated into laboratory tests that measure the concentrating ability of the kidney. Throughout the years, various methods have been used to produce water deprivation, including the Fishberg and Mosenthal concentration tests, which measured specific gravity. In the Fishberg test, patients were deprived of fluids for 24 hours before measuring specific gravity. The Mosenthal test compared Patient A Patient B Water (1 glass) Water (4 glasses) Glomerulus Glomerulus Ultrafiltrate 120 mL Water 300 mg Solute Reabsorption Urine 1 mL Water 200 mg Solute Specific gravity 1.015 Ultrafiltrate 119 mL Water 100 mg Solute Osmolality As will be discussed in Chapter 4, osmolality measures only the number of particles in a solution, whereas specific gravity is influenced by the number and density (molecular weight) of the particles. Renal concentration is concerned with small particles, primarily sodium and chloride molecules. Largemolecular-weight molecules such as glucose and urea do not contribute to the evaluation of renal concentration. Therefore osmolality is performed for a more accurate evaluation of renal concentrating ability. 120 mL Water 300 mg Solute Reabsorption Urine 10 mL Water 200 mg Solute Specific gravity 1.005 110 mL Water 100 mg Solute Figure 3–14 The effect of hydration on renal concentration. Notice the decreased specific gravity in the morehydrated Patient B. 3920_Ch03_039-057 23/01/14 10:10 AM Page 51 Chapter 3 | Renal Function Polyuria Urine osmolality <200 mOsm, Urine:Serum 1:1 Fluid Restriction Urine osmolality <400 mOsm, Urine:Serum 1:1 ADH Challenge Urine osmolality >800 mOsm, Urine:Serum 3:1 Urine osmolality <400 mOsm, Urine:Serum 1:1 Neurogenic diabetes insipidus Nephrogenic diabetes insipidus Figure 3–15 Differentiation of neurogenic and nephrogenic diabetes insipidus. Freezing Point Osmometers Measurement of freezing point depression was the first principle incorporated into clinical osmometers, and many instruments employing this technique are available. These osmometers determine the freezing point of a solution by supercooling a measured amount of sample to approximately 27°C. The supercooled sample is vibrated to produce crystallization of water in the solution. The heat of fusion produced by the crystallizing water temporarily raises the temperature of the solution to its freezing point. A temperature-sensitive probe called a thermistor, in which resistance decreases as temperature increases, measures this temperature increase, which corresponds to the freezing point of the solution, and the information is converted into milliosmoles. Conversion is made possible by the fact that 1 mol (1000 mOsm) of a nonionizing substance dissolved in 1 kg of water is known to lower the freezing point 1.86°C. Therefore, by comparing the freezing point depression of an unknown solution with that of a known molal solution, the osmolarity of the unknown solution can be calculated. Clinical osmometers use solutions of known NaCl concentration as their reference standards because a solution of partially ionized substances is more representative of urine and plasma composition. Vapor Pressure Osmometers The other instrument used in clinical osmometry is called the vapor pressure osmometer. The actual measurement performed, however, is that of the dew point (temperature at which water vapor condenses to a liquid). The depression of dew point temperature by solute parallels the decrease in vapor pressure, thereby providing a measure of this colligative property. Samples are absorbed into small filter paper disks that are placed in a sealed chamber containing a temperature-sensitive thermocoupler. The sample evaporates in the chamber, forming a vapor. When the temperature in the chamber is lowered, water condenses in the chamber and on the thermocoupler. The heat of condensation produced raises the temperature of 51 the thermocoupler to the dew point temperature. This dew point temperature is proportional to the vapor pressure from the evaporating sample. Temperatures are compared with those of the NaCl standards and converted into milliosmoles. The vapor pressure osmometer uses microsamples of less than 0.01 mL; therefore, care must be taken to prevent any evaporation of the sample prior to testing. Correlation studies have shown more variation with vapor pressure osmometers, stressing the necessity of careful technique. Technical Factors Factors to consider because of their influence on true osmolarity readings include lipemic serum, lactic acid, and volatile substances, such as ethanol, in the specimen. In lipemic serum, the serum water displacement by insoluble lipids produces erroneous results with both vapor pressure and freezing point osmometers. Falsely elevated values owing to lactic acid formation also occur with both methods if serum samples are not separated or refrigerated within 20 minutes. Vapor pressure osmometers do not detect the presence of volatile substances, such as alcohol, as they become part of the solvent phase; however, measurements performed on similar specimens using freezing point osmometers will be elevated. Clinical Significance Major clinical uses of osmolarity include initially evaluating renal concentrating ability, monitoring the course of renal disease, monitoring fluid and electrolyte therapy, establishing the differential diagnosis of hypernatremia and hyponatremia, and evaluating the secretion of and renal response to ADH. These evaluations may require determination of serum in addition to urine osmolarity. Reference serum osmolality values are from 275 to 300 mOsm. Reference values for urine osmolality are difficult to establish, because factors such as fluid intake and exercise can greatly influence the urine concentration. Values can range from 50 to 1400 mOsm.2 Determining the ratio of urine to serum osmolality can provide a more accurate evaluation. Under normal random conditions, the ratio of urine to serum osmolality should be at least 1:1; after controlled fluid intake, it should reach 3:1 (see Fig. 3–15). The ratio of urine to serum osmolality, in conjunction with procedures such as controlled fluid intake and injection of ADH, is used to differentiate whether diabetes insipidus is caused by decreased ADH production or inability of the renal tubules to respond to ADH. Failure to achieve a ratio of 3:1 after injecting ADH indicates that the collecting duct does not have functional ADH receptors. In contrast, if concentration takes place after ADH injection, an inability to produce TECHNICAL TIP Vapor pressure osmometers are used primarily to analyze serum and sweat microsamples for disorders not related to renal function, such as cystic fibrosis. They are used primarily in the chemistry department. 3920_Ch03_039-057 23/01/14 10:10 AM Page 52 52 Part One | Background adequate ADH is indicated. Tests to measure the ADH concentration in plasma and urine directly are available for difficult diagnostic cases.9 Free Water Clearance The ratio of urine to serum osmolarity can be further expanded by performing the analyses using water deprivation and a timed urine specimen and calculating the free water clearance. The free water clearance is determined by first calculating the osmolar clearance using the standard clearance formula: Cosm = Uosm × V Posm and then subtracting the osmolar clearance value from the urine volume in mL/min. EXAMPLE Using a urine osmolality of 600 mOsm (U), a urine volume of 2 mL/min (V), and a plasma osmolality of 300 mOsm (P), calculate the free water clearance: Cosm = 600 (U) × 2 (V) = 4.0 mL/min 300 (P) CH2O = 2 (V) – 4.0 (Cosm) = –2.0 (free water clearance) Calculating osmolar clearance indicates how much water must be cleared each minute to produce a urine with the same osmolality as the plasma. The ultrafiltrate contains the same osmolality as the plasma; therefore, the osmotic differences in the urine are the result of renal concentrating and diluting mechanisms. By comparing the osmolar clearance with the actual urine volume excreted per minute, it can be determined whether the water being excreted is more or less than the amount needed to maintain an osmolality the same as that of the ultrafiltrate. The above calculation shows a free water clearance of –2.0, indicating that less than the necessary amount of water is being excreted, a possible state of dehydration. If the value had been 0, no renal concentration or dilution would be taking place; likewise, if the value had been +2.0, excess water would have been excreted. The calculation of the free water clearance is used to determine the ability of the kidney to respond to the state of body hydration. Tubular Secretion and Renal Blood Flow Tests Tests to measure tubular secretion of nonfiltered substances and renal blood flow are closely related in that total renal blood flow through the nephron must be measured by a substance that is secreted rather than filtered through the glomerulus. Impaired tubular secretory ability or inadequate presentation of the substance to the capillaries owing to decreased renal blood flow may cause an abnormal result. Therefore, an understanding of the principles and limitations of the tests and correlation with other clinical data is important in test interpretation. The test most commonly associated with tubular secretion and renal blood flow is the p-aminohippuric acid (PAH) test. PAH Test To measure the exact amount of blood flowing through the kidney, it is necessary to use a substance that is completely removed from the blood (plasma) each time it comes in contact with functional renal tissue. The principle is the same as in the clearance test for glomerular filtration. However, to ensure measurement of the blood flow through the entire nephron, the substance must be removed from the blood primarily in the peritubular capillaries rather than being removed when the blood reaches the glomerulus. Although it has the disadvantage of being exogenous, the chemical PAH meets the criteria needed to measure renal blood flow. This nontoxic substance is loosely bound to plasma proteins, which permits its complete removal as the blood passes through the peritubular capillaries. Except for a small amount of PAH contained in plasma that does not come in contact with functional renal tissue, all the plasma PAH is secreted by the proximal convoluted tubule. Therefore, the volume of plasma flowing through the kidneys determines the amount of PAH excreted in the urine. The standard clearance formula can be used to calculate the effective renal plasma flow: CPAH (mL/min) = U (mg/dL PAH) × V (mL/min urine) P (mg/dL PAH) Based on normal hematocrit readings, reference values for the effective renal plasma flow range from 600 to 700 mL/min, making the average renal blood flow about 1200 mL/min. The actual measurement is renal plasma flow rather than renal blood flow, because the PAH is contained only in the plasma portion of the blood. Also, the term “effective” is included because approximately 8% of the renal blood flow does not come into contact with the functional renal tissue. The amount of PAH infused by IV must be monitored carefully to ensure accurate results; therefore, the test is usually performed by specialized renal laboratories. Nuclear medicine procedures using radioactive hippurate can determine renal blood flow by measuring the plasma disappearance of a single radioactive injection and at the same time provide visualization of the blood flowing through the kidneys.9 Titratable Acidity and Urinary Ammonia The ability of the kidney to produce an acid urine depends on the tubular secretion of hydrogen ions and production and secretion of ammonia by the cells of the distal convoluted tubule. A normal person excretes approximately 70 mEq/day of acid in the form of titratable acid (H+), hydrogen phosphate ions (H2PO4–), or ammonium ions (NH4+). In normal persons, a diurnal variation in urine acidity consisting of alkaline tides appears shortly after arising and postprandially at approximately 2 p.m. and 8 p.m. The lowest pH is found at night. 3920_Ch03_039-057 23/01/14 10:10 AM Page 53 Chapter 3 | Renal Function HISTORICAL NOTE the ammonium concentration can be calculated as the difference between the titratable acidity and the total acidity. Phenolsulfonphthalein Test Historically, excretion of the dye phenolsulfonphthalein (PSP) was used to evaluate these functions. Standardization and interpretation of PSP results are difficult, however, because of interference by medications, elevated waste products in patients’ serum, the necessity to obtain several very accurately timed urine specimens, and the possibility of producing anaphylactic shock. Therefore, the PSP test is not currently performed. The inability to produce an acid urine in the presence of metabolic acidosis is called renal tubular acidosis. This condition may result from impaired tubular secretion of hydrogen ions associated with the proximal convoluted tubule or defects in ammonia secretion associated with the distal convoluted tubule. Urine pH, titratable acidity, and urinary ammonia measurements can be used to determine the defective function. The tests can be run simultaneously on either fresh or toluenepreserved urine specimens collected at 2-hour intervals from patients who have been primed with an acid load consisting of oral ammonium chloride. By titrating the amount of free H+ (titratable acidity) and then the total acidity of the specimen, 53 Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Berger, A: Renal function and how to assess it. Brit J Med 321:1444, 2000. 2. Pincus, MR, Preuss, HG, and Henry, JB: Evaluation of renal function and water, electrolyte and acid-base balance. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 1996. 3. Levey, AS, et al: A new equation to estimate glomerular filtration rate. Ann Intern Med 150(9):601–612, 2009. 4. Laterza, OE, Price, CP, and Scott, MG: Cystatin C: An improved estimator of glomerular filtration rate? Clin Chem 48(5): 699–707, 2002. 5. Tan, GS, et al: Clinical usefulness of cystatin C for the estimation of glomerular filtration rate in type 1 diabetes. Crit Care 9(2):139–143, 2005. 6. Inker, LA: Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 367:20–29, 2012. 7. Foley, K: Beta 2 microglobulin: a facultative marker. Advance for MLP, Sept 30, 2008, p 13. 8. Chachati, A, et al: Rapid method for the measurement of differential renal function: Validation. J Nucl Med 28(5): 829–836, 1987. 9. Daves, BB, and Zenser, TV: Evaluation of renal concentrating and diluting ability. Clin Lab Med 13(1):131–134, 1993. Study Questions 1. The type of nephron responsible for renal concentration is the: A. Cortical B. Juxtaglomerular 2. The function of the peritubular capillaries is: A. Reabsorption B. Filtration C. Secretion D. Both A and C 3. Blood flows through the nephron in the following order: A. Efferent arteriole, peritubular capillaries, vasa recta, afferent arteriole B. Peritubular capillaries, afferent arteriole, vasa recta, efferent arteriole C. Afferent arteriole, peritubular capillaries, vasa recta, efferent arteriole D. Efferent arteriole, vasa recta, peritubular capillaries, afferent arteriole 4. Filtration of protein is prevented in the glomerulus by: A. Hydrostatic pressure B. Oncotic pressure C. Renin D. The glomerular filtration barrier 5. The renin-angiotensin-aldosterone system is responsible for all of the following except: A. Vasoconstriction of the afferent arteriole B. Vasoconstriction of the efferent arteriole C. Reabsorbing sodium D. Releasing aldosterone 6. The primary chemical affected by the renin-angiotensinaldosterone system is: A. Chloride B. Sodium C. Potassium D. Hydrogen 3920_Ch03_039-057 23/01/14 10:10 AM Page 54 54 Part One | Background 7. Secretion of renin is stimulated by: A. Juxtaglomerular cells 14. ADH regulates the final urine concentration by controlling: B. Angiotensin I and II A. Active reabsorption of sodium C. Macula densa cells B. Tubular permeability D. Circulating angiotensin-converting enzyme 8. The hormone aldosterone is responsible for: A. Hydrogen ion secretion B. Potassium secretion C. Chloride retention D. Sodium retention 9. The fluid leaving the glomerulus has a specific gravity of: A. 1.005 B. 1.010 C. 1.015 D. 1.020 10. For active transport to occur, a chemical: A. Must combine with a carrier protein to create electrochemical energy B. Must be filtered through the proximal convoluted tubule C. Must be in higher concentration in the filtrate than in the blood D. Must be in higher concentration in the blood than in the filtrate 11. Which of the tubules is impermeable to water? A. Proximal convoluted tubule B. Descending loop of Henle C. Ascending loop of Henle D. Distal convoluted tubule 12. Glucose will appear in the urine when the: A. Blood level of glucose is 200 mg/dL B. Tm for glucose is reached C. Renal threshold for glucose is exceeded D. All of the above 13. Concentration of the tubular filtrate by the countercurrent mechanism depends on all of the following except: A. High salt concentration in the medulla B. Water-impermeable walls of the ascending loop of Henle C. Passive reabsorption of urea D. Passive reabsorption of chloride 15. Decreased production of ADH: A. Produces a low urine volume B. Produces a high urine volume C. Increases ammonia excretion D. Affects active transport of sodium 16. Bicarbonate ions filtered by the glomerulus are returned to the blood: A. In the proximal convoluted tubule B. Combined with hydrogen ions C. By tubular secretion D. All of the above 17. If ammonia is not produced by the distal convoluted tubule, the urine pH will be: A. Acidic B. Basic 18. Place the appropriate letter in front of the following clearance substances: A. Exogenous B. Endogenous ____ beta2-microglobulin ____ creatinine ____ cystatin C ____ 125I-iothalmate 19. The largest source of error in creatinine clearance tests is: A. Secretion of creatinine B. Improperly timed urine specimens C. Refrigeration of the urine D. Time of collecting blood sample 20. Given the following information, calculate the creatinine clearance: 24-hour urine volume: 1000 mL; serum creatinine: 2.0 mg/dL; urine creatinine: 200 mg/dL 21. Clearance tests used to determine the glomerular filtration rate must measure substances that are: A. Not filtered by the glomerulus C. Reabsorption of sodium and chloride from the ascending loop of Henle B. Completely reabsorbed by the proximal convoluted tubule D. Reabsorption of water in the descending loop of Henle D. Neither reabsorbed or secreted by the tubules C. Secreted in the distal convoluted tubule 3920_Ch03_039-057 23/01/14 10:10 AM Page 55 Chapter 3 | Renal Function 22. Performing a clearance test using radionucleotides: A. Eliminates the need to collect urine 29. After controlled fluid intake, the urine-to-serum osmolarity ratio should be at least: B. Does not require an infusion A. 1:1 C. Provides visualization of the filtration B. 2:1 D. Both A and C 23. Variables that are included in the MDRD-IDSM estimated creatinine clearance calculations include all of the following except: A. Serum creatinine B. Weight C. Age D. Gender 24. An advantage to using cystatin C to monitor GFR is that: A. It does not require urine collection B. It is not secreted by the tubules C. It can be measured by immunoassay D. All of the above 25. Solute dissolved in solvent will: A. Raise the vapor pressure B. Lower the boiling point C. Decrease the osmotic pressure D. Lower the freezing point 26. Substances that may interfere with freezing point measurement of urine and serum osmolarity include all of the following except: A. Ethanol B. Lactic acid C. Sodium D. Lipids 27. Clinical osmometers use NaCl as a reference solution because: A. 1 g molecular weight of NaCl will lower the freezing point 1.86oC B. NaCl is readily frozen C. NaCl is partially ionized similar to the composition of urine D. 1 g equivalent weight of NaCl will raise the freezing point 1.86oC 28. The normal serum osmolarity is: A. 50 to 100 mOsm C. 3:1 D. 4:1 30. Calculate the free water clearance from the following results: urine volume in 6 hours: 720 mL; urine osmolarity: 225 mOsm; plasma osmolarity: 300 mOsm 31. To provide an accurate measure of renal blood flow, a test substance should be completely: A. Filtered by the glomerulus B. Reabsorbed by the tubules C. Secreted when it reaches the distal convoluted tubule D. Cleared on each contact with functional renal tissue 32. Given the following data, calculate the effective renal plasma flow: urine volume in 2 hours: 240 mL; urine PAH: 150 mg/dL; plasma PAH: 0.5 mg/dL 33. Renal tubular acidosis can be caused by the: A. Production of excessively acidic urine due to increased filtration of hydrogen ions B. Production of excessively acidic urine due to increased secretion of hydrogen ions C. Inability to produce an acidic urine due to impaired production of ammonia D. Inability to produce an acidic urine due to increased production of ammonia 34. Tests performed to detect renal tubular acidosis after administering an ammonium chloride load include all of the following except: A. Urine ammonia B. Arterial pH B. 275 to 300 mOsm C. Urine pH C. 400 to 500 mOsm D. Titratable acidity D. 3 times the urine osmolarity 55 3920_Ch03_039-057 23/01/14 10:10 AM Page 56 56 Part One | Background Case Studies and Clinical Situations 1. A 44-year-old man diagnosed with acute tubular necrosis has a blood urea nitrogen of 60 mg/dL and a blood glucose level of 100 mg/dL. A 2+ urine glucose is also reported. a. State the renal threshold for glucose. b. What is the significance of the positive urine glucose and normal blood glucose? 2. A patient develops a sudden drop in blood pressure. a. Diagram the reactions that take place to ensure adequate blood pressure within the nephrons. b. How do these reactions increase blood volume? c. When blood pressure returns to normal, how does the kidney respond? 3. A physician would like to prescribe a nephrotoxic antibiotic for a 60-year-old Caucasian man. The patient has a serum creatinine level of 1.5 mg/dL. a. How can the physician determine whether it is safe to prescribe this medication before the patient leaves the office? b. State two additional blood tests that the physician could use to continue monitoring this patient. c. If the patient has a history of prostate malignancy, would both of these methods provide reliable results? Explain your answer. 4. A laboratory is obtaining erratic serum osmolarity results on a patient who is being monitored at 6 a.m., 12 p.m., 6 p.m., and 12 a.m. Osmolarities are not performed on the night shift; therefore, the midnight specimen is run at the same time as the 6 a.m. specimen. a. What two reasons could account for these discrepancies? b. What substance is causing the erratic results? c. If a friend were secretly bringing the patient a pint of whiskey every night, would this affect the results? Explain your answer. 5. Following overnight (6 p.m. to 8 a.m.) fluid deprivation, the urine-to-serum osmolarity ratio in a patient who is exhibiting polyuria and polydipsia is 1:1. The ratio remains the same when a second specimen is tested at 10 a.m. ADH is then administered subcutaneously to the patient, and the fluid deprivation is continued until 2 p.m., when another specimen is tested. a. What disorder do these symptoms and initial laboratory results indicate? b. If the urine-to-serum osmolarity ratio on the 2 p.m. specimen is 3:1, what is the underlying cause of the patient’s disorder? c. If the urine-to-serum osmolarity ratio on the 2 p.m. specimen remains 1:1, what is the underlying cause of the patient’s disorder? 3920_Ch03_039-057 23/01/14 10:10 AM Page 57 3920_Ch04_058-070 23/01/14 10:55 AM Page 58 PART T WO Urinalysis Chapter 4: Physical Examination of Urine Chapter 5: Chemical Examination of Urine Chapter 6: Microscopic Examination of Urine Chapter 7: Renal Disease Chapter 8: Urine Screening for Metabolic Disorders 3920_Ch04_058-070 23/01/14 10:55 AM Page 59 CHAPTER 4 Physical Examination of Urine LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 4-1 List the common terminology used to report normal urine color. 4-10 List three pathologic and four nonpathologic causes of cloudy urine. 4-2 Discuss the relationship of urochrome to normal urine color. 4-11 Define specific gravity, and tell why this measurement can be significant in the routine analysis. 4-3 State how the presence of bilirubin, biliverdin, uroerythrin, and urobilin in a specimen may be suspected. 4-12 Describe the principles of the refractometer, reagent strip, and osmolality for determining specific gravity. 4-4 Discuss the significance of cloudy red urine and clear red urine. 4-13 Given the concentration of glucose and protein in a specimen, calculate the correction needed to compensate for these high-molecular-weight substances in the refractometer specific gravity reading. 4-5 Name two pathologic causes of black or brown urine. 4-6 Discuss the significance of phenazopyridine in a specimen. 4-7 State the clinical significance of urine clarity. 4-8 List the common terminology used to report clarity. 4-9 Describe the appearance and discuss the significance of amorphous phosphates and amorphous urates in freshly voided urine. 4-14 Name two nonpathogenic causes of abnormally high specific gravity readings using a refractometer. 4-15 Describe the advantages of measuring specific gravity using a reagent strip and osmolality. 4-16 State possible causes of abnormal urine odor. KEY TERMS Clarity Isosthenuric Urobilin Hypersthenuric Refractometry Urochrome Hyposthenuric Specific gravity Uroerythrin 3920_Ch04_058-070 23/01/14 10:55 AM Page 60 60 Part Two | Urinalysis The physical examination of urine includes the determination of the urine color, clarity, and specific gravity. As mentioned in Chapter 2, early physicians based many medical decisions on the color and clarity of urine. Today, observation of these characteristics provides preliminary information concerning disorders such as glomerular bleeding, liver disease, inborn errors of metabolism, and urinary tract infection. Measurement of specific gravity aids in the evaluation of renal tubular function. The results of the physical portion of the urinalysis also can be used to confirm or to explain findings in the chemical and microscopic areas of urinalysis. Color The color of urine varies from almost colorless to black. These variations may be due to normal metabolic functions, physical activity, ingested materials, or pathologic conditions. A noticeable change in urine color is often the reason that a patient Table 4–1 seeks medical advice; it then becomes the responsibility of the laboratory to determine whether this color change is normal or pathologic. The more common normal and pathologic correlations of urine colors are summarized in Table 4–1. Normal Urine Color Terminology used to describe the color of normal urine may differ slightly among laboratories but should be consistent within each laboratory. Common descriptions include pale yellow, yellow, and dark yellow. Care should be taken to examine the specimen under a good light source, looking down through the container against a white background. The yellow color of urine is caused by the presence of a pigment, which Thudichum named urochrome in 1864. Urochrome is a product of endogenous metabolism, and under normal conditions the body produces it at a constant rate. The actual amount of urochrome produced is dependent on the body’s metabolic state, with increased amounts produced in thyroid conditions and fasting Laboratory Correlation of Urine Color1 Color Cause Clinical/Laboratory Correlations Colorless Recent fluid consumption Polyuria or diabetes insipidus Diabetes mellitus Dilute random specimen Concentrated specimen Commonly observed with random specimens Pale yellow Dark yellow B complex vitamins Dehydration Bilirubin Orange-yellow Yellow-green Green Blue-green Pink Red Acriflavine Nitrofurantoin Phenazopyridine (Pyridium) Phenindione Bilirubin oxidized to biliverdin Pseudomonas infection Amitriptyline Methocarbamol (Robaxin) Clorets Indican Methylene blue Phenol RBCs Hemoglobin Increased 24-hour volume and low specific gravity Elevated specific gravity and positive glucose test result Recent fluid consumption May be normal after strenuous exercise or in first morning specimen Fever or burns Yellow foam when shaken and positive chemical test results for bilirubin Negative bile test results and possible green fluorescence Antibiotic administered for urinary tract infections Drug commonly administered for urinary tract infections Anticoagulant, orange in alkaline urine, colorless in acid urine Colored foam in acidic urine and false-negative chemical test results for bilirubin Positive urine culture Antidepressant Muscle relaxant, may be green-brown None Bacterial infections, intestinal disorders Fistulas When oxidized Cloudy urine with positive chemical test results for blood and RBCs visible microscopically Clear urine with positive chemical test results for blood; intravascular hemolysis 3920_Ch04_058-070 23/01/14 10:55 AM Page 61 Chapter 4 | Physical Examination of Urine 61 Table 4–1 Color Port wine Red-brown Brown Black Laboratory Correlation of Urine Color1—cont’d Cause Clinical/Laboratory Correlations Myoglobin Clear urine with positive chemical test results for blood; muscle damage Alkaline urine of genetically susceptible persons Tuberculosis medication Cloudy specimen with RBCs, mucus, and clots Negative test for blood, may require additional testing Seen in acidic urine after standing; positive chemical test result for blood Beets Rifampin Menstrual contamination Porphyrins RBCs oxidized to methemoglobin Myoglobin Homogentisic acid (alkaptonuria) Malignant melanoma Melanin or melanogen Phenol derivatives Argyrol (antiseptic) Methyldopa or levodopa Metronidazole (Flagyl) states.2 Urochrome also increases in urine that stands at room temperature.3 Because urochrome is excreted at a constant rate, the intensity of the yellow color in a fresh urine specimen can give a rough estimate of urine concentration. A dilute urine will be pale yellow and a concentrated specimen will be dark yellow. Remember that, owing to variations in the body’s state of hydration, these differences in the yellow color of urine can be normal. Two additional pigments, uroerythrin and urobilin, are also present in the urine in much smaller quantities, and contribute little to the color of normal, fresh urine. The presence of uroerythrin, a pink pigment, is most evident in specimens that have been refrigerated, resulting in the precipitation of amorphous urates. Uroerythrin attaches to the urates, producing a pink color to the sediment. Urobilin, an oxidation product of the normal urinary constituent urobilinogen, imparts an orange-brown color to urine that is not fresh. Abnormal Urine Color As can be seen in Table 4–1, abnormal urine colors are as numerous as their causes. Certain colors, however, are seen more frequently and have a greater clinical significance than do others. Dark Yellow/Amber/Orange Dark yellow or amber urine may not always signify a normal concentrated urine but can be caused by the presence of the abnormal pigment bilirubin. If bilirubin is present, it will be detected during the chemical examination; however, its Seen in alkaline urine after standing; specific tests are available Urine darkens on standing and reacts with nitroprusside and ferric chloride Interfere with copper reduction tests Color disappears with ferric chloride Antihypertensive Darkens on standing, intestinal and vaginal infections presence is suspected if yellow foam appears when the specimen is shaken. Normal urine produces only a small amount of rapidly disappearing foam when shaken, and a large amount of white foam indicates an increased concentration of protein. A urine specimen that contains bilirubin may also contain hepatitis virus, reinforcing the need to follow standard precautions. The photo-oxidation of large amounts of excreted urobilinogen to urobilin also produces a yellow-orange urine; however, yellow foam does not appear when the specimen is shaken. Photo-oxidation of bilirubin imparts a yellow-green color to the urine caused by the presence of biliverdin. Also frequently encountered in the urinalysis laboratory is the yellow-orange specimen caused by the administration of phenazopyridine (brand name Pyridium) or azo-gantrisin compounds to people who have urinary tract infections. This thick, orange pigment not only obscures the natural color of the specimen but also interferes with chemical tests that are based on color reactions. It is important to recognize the presence of phenazopyridine in a specimen so that laboratories can use alternative testing procedures. Specimens containing phenazopyridine produce a yellow foam when shaken, which could be mistaken for bilirubin. Red/Pink/Brown One of the most common causes of abnormal urine color is the presence of blood. Red is the usual color that blood produces in urine, but the color may range from pink to brown, depending on the amount of blood, the pH of the urine, and the length of contact. Red blood cells (RBCs) remaining in an acidic urine for several hours produce a brown 3920_Ch04_058-070 23/01/14 10:55 AM Page 62 62 Part Two | Urinalysis urine due to the oxidation of hemoglobin to methemoglobin. A fresh brown urine containing blood may also indicate glomerular bleeding resulting from the conversion of hemoglobin to methemoglobin.4 Besides RBCs, two other substances, hemoglobin and myoglobin, produce a red urine and result in a positive chemical test result for blood (Fig. 4–1). When RBCs are present, the urine is red and cloudy; however, if hemoglobin or myoglobin is present, the specimen is red and clear. Distinguishing between hemoglobinuria and myoglobinuria may be possible by examining the patient’s plasma. Hemoglobinuria resulting from the in vivo breakdown of RBCs is accompanied by red plasma. Breakdown of skeletal muscle produces myoglobin. Myoglobin is more rapidly cleared from the plasma than is hemoglobin and, therefore, does not affect the color of the plasma. Fresh urine containing myoglobin frequently exhibits a more reddish-brown color than does urine containing hemoglobin. The possibility of hemoglobinuria being produced from the in vitro lysis of RBCs also must be considered. Chemical tests to distinguish between hemoglobin and myoglobin are available (see Chapter 5). Urine specimens containing porphyrins also may appear red, resulting from the oxidation of porphobilinogen to porphyrins. They are often referred to as having the color of port wine. Nonpathogenic causes of red urine include menstrual contamination, ingestion of highly pigmented foods, and medications. In genetically susceptible persons, eating fresh beets causes a red color in alkaline urine.5 Ingestion of blackberries can produce a red color in acidic urine. Many medications, including rifampin, phenolphthalein, phenindione, and phenothiazines, produce red urine. Brown/Black Additional testing is recommended for urine specimens that turn brown or black on standing and have negative chemical test results for blood, inasmuch as they may contain melanin or homogentisic acid. Melanin is an oxidation product of the colorless pigment, melanogen, produced in excess when a malignant melanoma is present. Homogentisic acid, a metabolite of phenylalanine, imparts a black color to alkaline urine from persons with the inborn-error of metabolism, called Red urine Clear Hemoglobinuria Red plasma Cloudy Myoglobinuria Red blood cells present (Hematuria) Clear plasma Figure 4–1 Differentiation of red urine testing chemically positive for blood. alkaptonuria. These conditions are discussed in Chapter 7. Medications producing brown/black urines include levodopa, methyldopa, phenol derivatives, and metronidazole (Flagyl). Blue/Green Pathogenic causes of blue/green urine are limited to bacterial infections, including urinary tract infection by Pseudomonas species and intestinal tract infections resulting in increased urinary indican. Ingestion of breath deodorizers (Clorets) can result in a green urine.6 The medications methocarbamol (Robaxin), methylene blue, and amitriptyline (Elavil) may cause blue urine. Observation of specimen collection bags from hospitalized patients frequently reveals abnormally colored urine. This may signify either a pathologic condition that requires the urine to stand for a period of time before color development, or the presence of medications. Phenol derivatives found in certain intravenous medications produce green urine on oxidation.7 A purple staining may occur in catheter bags and is caused by indican in the urine or a bacterial infection, frequently caused by Klebsiella or Providencia species.8 Clarity “Clarity” is a general term that refers to the transparency or turbidity of a urine specimen. In routine urinalysis, clarity is determined in the same manner that ancient physicians used: by visually examining the mixed specimen while holding it in front of a light source. The specimen should, of course, be in a clear container. Color and clarity are routinely determined at the same time. Common terminology used to report clarity includes clear, hazy, cloudy, turbid, and milky. As discussed previously under the section on urine color, terminology should be consistent within a laboratory. A description of urine clarity reporting is presented in Table 4–2. Normal Clarity Freshly voided normal urine is usually clear, particularly if it is a midstream clean-catch specimen. Precipitation of amorphous phosphates and carbonates may cause a white cloudiness. Table 4–2 Urine Clarity Clarity Term Clear Hazy No visible particulates, transparent Few particulates, print easily seen through urine Many particulates, print blurred through urine Print cannot be seen through urine May precipitate or be clotted Cloudy Turbid Milky 3920_Ch04_058-070 23/01/14 10:55 AM Page 63 Chapter 4 | Physical Examination of Urine 63 PROCEDURE 4-1 Urine Color and Clarity Procedure 1. Evaluate an adequate volume of specimen. 2. Use a well-mixed specimen. 3. View the urine through a clear container. 4. View the urine against a white background using adequate room lighting. 5. Maintain adequate room lighting 6. Evaluate a consistent volume of urine • Determine the urine color. • Describe the urine clarity (Table 4–2). Nonpathologic Turbidity The presence of squamous epithelial cells and mucus, particularly in specimens from women, can result in a hazy but normal urine. Specimens that are allowed to stand or are refrigerated also may develop turbidity that is nonpathologic. As discussed in Chapter 2, improper preservation of a specimen results in bacterial growth; this increases specimen turbidity but is not representative of the actual specimen. Refrigerated specimens frequently develop a thick turbidity caused by the precipitation of amorphous phosphates, carbonates, and urates. Amorphous phosphates and carbonates produce a white precipitate in urine with an alkaline pH, whereas amorphous urates produce a precipitate in acidic urine that resembles pink brick dust due to the presence of uroerythrin. Additional nonpathologic causes of urine turbidity include semen, fecal contamination, radiographic contrast media, talcum powder, and vaginal creams (Table 4–3). Pathologic Turbidity The most commonly encountered pathologic causes of turbidity in a fresh specimen are RBCs, white blood cells (WBCs), and bacteria caused by infection or a systemic organ disorder. Other, less frequently encountered causes of pathologic turbidity include abnormal amounts of nonsquamous Table 4–3 Nonpathologic Causes of Urine Turbidity Squamous epithelial cells Mucus Amorphous phosphates, carbonates, urates Semen, spermatozoa Fecal contamination Radiographic contrast media Talcum powder Vaginal creams epithelial cells, yeast, abnormal crystals, lymph fluid, and lipids (Table 4–4). The clarity of a urine specimen certainly provides a key to the microscopic examination results, because the amount of turbidity should correspond with the amount of material observed under the microscope. Clear urine is not always normal. However, with the increased sensitivity of the routine chemical tests, most abnormalities in clear urine will be detected prior to the microscopic analysis. Current criteria used to determine the necessity of performing a microscopic examination on all urine specimens include both clarity and chemical tests for RBCs, WBCs, bacteria, and protein. Specific Gravity As discussed in Chapter 3, the kidney’s ability to concentrate the glomerular filtrate by selectively reabsorbing essential chemicals and water from the glomerular filtrate is one of the kidney’s most important functions. The evaluation of urine concentration is included in the routine urinalysis by measuring the specific gravity of the specimen. Including specific gravity in the routine urinalysis provides an additional function, which is to determine whether specimen concentration is adequate to ensure the accuracy of chemical tests. The specific gravity of the plasma filtrate entering the glomerulus is 1.010. The term isosthenuric is used to describe urine with a specific gravity of 1.010. Specimens below 1.010 are hyposthenuric, and those above 1.010 are hypersthenuric. One would expect urine that has been concentrated by the kidneys to be hypersthenuric, but this is not always true. Normal random specimens may range from approximately 1.002 to 1.035, depending on the patient’s amount of hydration. Specimens measuring lower than 1.002 probably are not urine. Most random specimens fall between 1.015 and 1.030. Specific gravity is defined as the density of a solution compared with the density of a similar volume of distilled water (SG 1.000) at a similar temperature. Because urine is actually water that contains dissolved chemicals, the specific gravity of urine is a measure of the density of the dissolved chemicals in the specimen. As a measure of specimen density, specific gravity is influenced not only by the number of particles present but also by their size. Therefore, large molecules contribute more to the reading than do the small molecules. This may require the need to correct for the presence of substances that are not normally seen in urine such as glucose and protein in the specimen. Currently the only method in use in routine Table 4–4 Pathologic Causes of Urine Turbidity RBCs WBCs Nonsquamous epithelial cells Abnormal crystals Bacteria Yeast Lymph fluid Lipids 3920_Ch04_058-070 23/01/14 10:55 AM Page 64 64 Part Two | Urinalysis urinalysis that requires correcting is the refractometer. The other two methods in use are chemical reagent strips and osmolality. The principles behind current specific gravity measurement techniques are presented in Box 4–1. Refractometer Refractometry determines the concentration of dissolved particles in a specimen by measuring refractive index. Refractive index is a comparison of the velocity of light in air with the velocity of light in a solution. The concentration of dissolved particles present in the solution determines the velocity and angle at which light passes through a solution. Clinical refractometers make use of these principles of light by using a prism to direct a specific (monochromatic) wavelength of daylight against a manufacturer-calibrated specific gravity scale. The concentration of the specimen determines the angle at which the light beam enters the prism. Therefore, the specific gravity scale is calibrated in terms of the angles at which light passes through the specimen. The refractometer provides the distinct advantage of determining specific gravity using a small volume of specimen (one or two drops). Temperature corrections are not necessary because the light beam passes through a temperaturecompensating liquid prior to being directed at the specific gravity scale. Temperature is compensated between 15°C and 38°C. Corrections for glucose and protein must be calculated by subtracting 0.003 for each gram of protein present and 0.004 for each gram of glucose present. The amount of protein or glucose present can be determined from the chemical reagent strip tests. When using the refractometer, a drop of urine is placed on the prism, the instrument is focused at a good light source, and the reading is taken directly from the specific gravity scale. The prism and its cover should be cleaned after each specimen is tested. Figure 4–2 illustrates the use of the refractometer. The refractometer is calibrated using distilled water that should read 1.000. If necessary, the instrument contains a zero setscrew to adjust the distilled water reading (Fig. 4–3). The calibration is further checked using 5% NaCl, which as shown in the refractometer conversion tables should read 1.022 ± 0.001, or 9% sucrose that should read 1.034 ± 0.001. Urine control samples representing low, medium, and high concentrations should also be run at the beginning of each shift. Calibration and control results are always recorded in the appropriate quality control records. Box 4-1 Current Urine Specific Gravity Measurements Method Principle Refractometry Refractive index Osmolality Changes in colligative properties by particle number Reagent strip pKa changes of a polyelectrolyte by ions present HISTORICAL NOTE Urinometry The urinometer consists of a weighted float attached to a scale that has been calibrated in terms of urine specific gravity. The weighted float displaces a volume of liquid equal to its weight and has been designed to sink to a level of 1.000 in distilled water. The additional mass provided by the dissolved substances in urine causes the float to displace a volume of urine smaller than that of distilled water. The level to which the urinometer sinks, as shown in the figure, represents the specimen’s mass or specific gravity. 0 10 0 20 10 30 20 40 30 50 40 50 Urinometers representing various specific gravity readings. Urinometry is less accurate than the other methods currently available and is not recommended by the Clinical and Laboratory Standards Institute (CLSI).9 EXAMPLE A specimen containing 1 g/dL protein and 1 g/dL glucose has a specific gravity reading of 1.030. Calculate the corrected reading. 1.030 – 0.003 (protein) = 1.027 – 0.004 (glucose) = 1.023 corrected specific gravity 3920_Ch04_058-070 23/01/14 10:55 AM Page 65 Chapter 4 | Physical Examination of Urine 65 O 1. Put one or two drops of sample on the prism. 2. Close the daylight plate gently. 1.050 X 3. The sample must spread all over the prism surface. 1.355 1.040 1.350 1.030 1.345 1.020 X 1.340 1.010 1.335 1.333 1.000 U.G. 20˚C nD 4. Look at the scale through the eyepiece. 5. Read the scale where the boundary line intercepts it. 6. Wipe the sample from the prism clean with a tissue paper and water. Figure 4–2 Steps in the use of the urine specific gravity refractometer. (Courtesy of NSG Precision Cells, Inc., 195G Central Ave., Farmingdale, NY, 11735.) Abnormally high results—above 1.040—are seen in patients who have recently undergone an intravenous pyelogram. This is caused by the excretion of the injected radiographic contrast media. Patients who are receiving dextran or other Calibration screw high-molecular-weight intravenous fluids (plasma expanders) also produce urine with an abnormally high specific gravity. Once the foreign substance has been cleared from the body, the specific gravity returns to normal. In these circumstances, urine concentration can be measured using the reagent strip chemical test or osmometry because they are not affected by these high-molecular-weight substances.10 Osmolality As stated previously, specific gravity depends on the number of particles present in a solution and the density of these particles; osmolality is affected only by the number of particles present. When evaluating renal concentration ability, the HISTORICAL NOTE Harmonic Oscillation Densitometry Figure 4–3 Calibration of the urine specific gravity refractometer. (Courtesy of NSG Precision Cells, Inc., 195G Central Ave., Farmingdale, NY, 11735.) Harmonic oscillation densitometry is based on the principle that the frequency of a sound wave entering a solution changes in proportion to the density of the solution. This technique was originally used in early automated urinalysis instruments. The addition of reagent strip analysis for specific gravity has replaced this technique in automated systems. 3920_Ch04_058-070 23/01/14 10:56 AM Page 66 66 Part Two | Urinalysis substances of interest are small molecules, primarily sodium (molecular weight 23) and chloride (molecular weight 35.5). However, urea (molecular weight 60), which is of no importance to this evaluation, will contribute more to the specific gravity than will the sodium and chloride molecules. Because all three molecules contribute equally to the osmolarity of the specimen, a more representative measure of renal concentrating ability can be obtained by measuring osmolarity (see Chapter 3). An osmole is defined as 1 g molecular weight of a substance divided by the number of particles into which it dissociates. A nonionizing substance such as glucose (molecular weight, 180) contains 180 g per osmole, whereas sodium chloride (NaCl) (molecular weight 58.5), if completely dissociated, contains 29.25 g per osmole. Just like molality and molarity, there are osmolality and osmolarity. An osmolal solution of glucose has 180 g of glucose dissolved in 1 kg of solvent. An osmolar solution of glucose has 180 g of glucose dissolved in 1 L of solvent. The unit of measure used in the clinical laboratory is the milliosmole (mOsm), because it is not practical when dealing with body fluids to use a measurement as large as the osmole (23 g of sodium per kilogram). The osmolarity of a solution can be determined by measuring a property that is mathematically related to the number of particles in the solution (colligative property) and comparing this value with the value obtained from the pure solvent. Solute dissolved in solvent causes the following changes in colligative properties: lower freezing point, higher boiling point, increased osmotic pressure, and lower vapor pressure (see Table 4–5). Because water is the solvent in urine the number of particles present in a sample can be determined by comparing a colligative property value of the sample with that of pure water. To measure osmolality in the urinalysis laboratory requires special equipment referred to as an osmometer and therefore an additional step in the routine urinalysis procedure. TECHNICAL TIP The term “molality” is most commonly used because the solute and the solvent are both expressed in the same units of measure. Table 4–5 Particle Changes to Colligative Properties Property Freezing Point Boiling Point Vapor Pressure Osmotic Pressure Normal Pure Water Point Effect of 1 Mole of Solute 0°C 100°C 2.38 mm/ Hg at 25°C 0 mm/Hg Lowered 1.86°C Raised 0.52°C Lowered 0.3 mm/ Hg at 25°C Increased 1.7 × 109 mm/Hg The A2O Advanced Automated Osmometer (Advanced Instruments, Inc., Two Technology Way, Norwood, MA 02062) uses freezing point depression to measure osmolality, providing a more automated method for measuring both urine and serum osmolality. (The principles and uses of the freezing point and vapor pressure osmometers currently in use in the clinical laboratory are covered in Chapter 3.) Additional information on osmometry can be found at http://www.aitests.com. On the home page click on AI University. A video can also be accessed there. Reagent Strip Specific Gravity The addition of a specific gravity testing area to urinalysis chemical reagent strips has provided a convenient way to perform the routine urinalysis by eliminating the need for an additional procedure. The reagent strip reaction is based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium. The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number of ions in the solution. The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH. Incorporation of the indicator bromthymol blue on the reagent pad measures the change in pH. As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow (1.030 [acid]). Readings can be made in 0.005 intervals by careful comparison with the color chart. A diagram of the specific gravity reaction is shown in Chapter 5, Figure 5.4. Odor Although it is seldom of clinical significance and is not a part of the routine urinalysis, urine odor is a noticeable physical property. Freshly voided urine has a faint aromatic odor. As the specimen stands, the odor of ammonia becomes more prominent. The breakdown of urea is responsible for the characteristic ammonia odor. Causes of unusual odors include bacterial infections, which cause a strong, unpleasant odor similar to ammonia, and diabetic ketones, which produce a sweet or fruity odor. A serious metabolic defect results in urine with a strong odor of maple syrup and is appropriately called maple syrup urine disease. This and other metabolic disorders with characteristic urine odors are discussed in Chapter 8. Ingestion of certain foods, including onions, garlic, and asparagus, can TECHNICAL TIP Because ions such as Na+, Cl–, and NH4+ are important in evaluating renal concentrating ability, the reagent strip method provides additional information and is not affected by nonionizing substances including urea, glucose, protein, and contaminating substances such as radiographic dye. 3920_Ch04_058-070 23/01/14 10:56 AM Page 67 Chapter 4 | Physical Examination of Urine 67 cause an unusual or pungent urine odor. Studies have shown that although everyone who eats asparagus produces an odor, only certain genetically predisposed people can smell the odor.11 Common causes of urine odors are summarized in Table 4–6. Table 4–6 Possible Causes of Urine Odor1 Odor Cause Aromatic Foul, ammonia-like Normal Bacterial decomposition, urinary tract infection Ketones (diabetes mellitus, starvation, vomiting) Maple syrup urine disease Phenylketonuria Tyrosinemia Isovaleric acidemia Methionine malabsorption Contamination Fruity, sweet Maple syrup Mousy Rancid Sweaty feet Cabbage Bleach References 1. Henry, JB, Lauzon, RB, and Schumann, GB: Basic examination of urine. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 1996. 2. Drabkin, DL: The normal pigment of urine: The relationship of urinary pigment output to diet and metabolism. J Biol Chem 75:443–479, 1927. 3. Ostow, M, and Philo, S: The chief urinary pigment: The relationship between the rate of excretion of the yellow pigment and the metabolic rate. Am J Med Sci 207:507–512, 1944. 4. Berman, L: When urine is red. JAMA 237:2753–2754, 1977. 5. Reimann, HA: Re: Red urine. JAMA 241(22):2380, 1979. 6. Evans, B: The greening of urine: Still another “Cloret sign.” N Engl J Med 300(4):202, 1979. 7. Bowling, P, Belliveau, RR, and Butler, TJ: Intravenous medications and green urine. JAMA 246(3):216, 1981. 8. Dealler, SF, et al: Purple urine bags. J Urol 142(3):769–770, 1989. 9. Clinical and Laboratory Standards Institute (Formerly NCCLS) Approved Guideline GP16-A3: Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline, ed 3, CLSI, Wayne, PA, 2009. 10. Smith, C, Arbogast, C, and Phillips, R: Effect of x-ray contrast media on results for relative density of urine. Clin Chem 19(4):730–731, 1983. 11. Mitchell, SC, et al: Odorous urine following asparagus ingestion in man. Experimenta 43(4):382–383, 1987. Log on to www.fadavis.com/strasinger for additional content related to this chapter. Study Questions 1. The concentration of a normal urine specimen can be estimated by which of the following? A. Color B. Clarity C. Foam D. Odor 2. The normal yellow color of urine is produced by: A. Bilirubin B. Hemoglobin C. Urobilinogen D. Urochrome 3. The presence of bilirubin in a urine specimen produces a: A. Yellow foam when shaken B. White foam when shaken C. Cloudy specimen D. Yellow-red specimen 4. A urine specimen containing melanin will appear: A. Pale pink B. Dark yellow C. Blue-green D. Black 5. Specimens that contain hemoglobin can be visually distinguished from those that contain RBCs because: A. Hemoglobin produces a clear, yellow specimen B. Hemoglobin produces a cloudy pink specimen C. RBCs produce a cloudy red specimen D. RBCs produce a clear red specimen 6. A patient with a viscous orange specimen may have been: A. Treated for a urinary tract infection B. Taking vitamin B pills C. Eating fresh carrots D. Taking antidepressants 3920_Ch04_058-070 23/01/14 10:56 AM Page 68 68 Part Two | Urinalysis 7. The presence of a pink precipitate in a refrigerated specimen is caused by: A. Hemoglobin B. Urobilin C. Uroerythrin D. Beets 8. Microscopic examination of a clear urine that produces a white precipitate after refrigeration will show: A. Amorphous urates B. Porphyrins C. Amorphous phosphates D. Yeast 9. The color of urine containing porphyrins will be: A. Yellow-brown B. Green C. Orange D. Port wine 10. Which of the following specific gravities would be most likely to correlate with a pale yellow urine? A. 1.005 B. 1.010 C. 1.020 D. 1.030 11. A urine specific gravity measured by refractometer is 1.029, and the temperature of the urine is 14°C. The specific gravity should be reported as: A. 1.023 B. 1.027 C. 1.029 D. 1.032 12. The principle of refractive index is to compare: A. Light velocity in solutions with light velocity in solids B. Light velocity in air with light velocity in solutions C. Light scattering by air with light scattering by solutions D. Light scattering by particles in solution 13. A correlation exists between a specific gravity by refractometer of 1.050 and a: A. 2+ glucose B. 2+ protein C. First morning specimen D. Radiographic dye infusion 14. A cloudy urine specimen turns black upon standing and has a specific gravity of 1.012. The major concern about this specimen would be: A. Color B. Turbidity C. Specific gravity D. All of the above 15. A specimen with a specific gravity of 1.035 would be considered: A. Isosthenuric B. Hyposthenuric C. Hypersthenuric D. Not urine 16. A specimen with a specific gravity of 1.001 would be considered: A. Hyposthenuric B. Not urine C. Hypersthenuric D. Isosthenuric 17. A strong odor of ammonia in a urine specimen could indicate: A. Ketones B. Normalcy C. Phenylketonuria D. An old specimen 18. The microscopic of a clear red urine is reported as many WBCs and epithelial cells. What does this suggest? A. Urinary tract infection B. Dilute random specimen C. Hematuria D. Possible mix-up of specimen and sediment 19. Which of the following would contribute the most to a urine osmolality? A. One osmole of glucose B. One osmole of urea C. One osmole of sodium chloride D. All contribute equally 20. Which of the following colligative properties is not stated correctly? A. The boiling pointing is raised by solute B. The freezing point is raised by solute C. The vapor pressure is lowered by solute D. The osmotic pressure is raised by solute 3920_Ch04_058-070 23/01/14 10:56 AM Page 69 Chapter 4 | Physical Examination of Urine 69 21. An osmole contains: A. One gram molecular weight of solute dissolved in one liter of solvent B. One gram molecular weight of solute dissolved in one kilogram of solvent C. Two gram molecular weights of solute dissolved in one liter of solvent D. Two gram molecular weights of solute dissolved in one kilogram of solvent 22. The unit of osmolality measured in the clinical laboratory is the: A. Osmole B. Milliosmole C. Molecular weight D. Ionic charge 23. In the reagent strip specific gravity reaction the polyelectrolyte: A. Combines with hydrogen ions in response to ion concentration B. Releases hydrogen ions in response to ion concentration C. Releases hydrogen ions in response to pH D. Combines with sodium ions in response to pH 24. Which of the following will react in the reagent strip specific gravity test? A. Glucose B. Radiographic dye C. Protein D. Chloride Case Studies and Clinical Situations 1. Given the following physical urinalysis results, determine additional urinalysis results that may be abnormal. a. A green specimen with a strong foul odor of ammonia b. A pale yellow urine with a specific gravity of 1.030 c. A dark yellow specimen with yellow foam d. A cloudy red urine c. If the specific gravity was also checked using osmometry, should the result agree with the laboratory or the urology clinic results? Why or why not? 3. State two pathologic causes of a clear red urine. a. State a method that could distinguish between the two causes that does not require laboratory testing. a. Correct the refractometer reading to account for the protein and glucose concentrations. What is the corrected specific gravity? 4. Mrs. Smith frequently shops at the farmer’s market near her home. She notices her urine has a red color and brings a sample to her physician. The specimen tests negative for blood. a. What is a probable cause of Mrs. Smith’s red urine? b. Mrs. Smith collects a specimen at the physician’s office. The color is yellow and the pH is 5.5. Is this consistent with the previous answer? Why or why not? b. Do the specific gravities correlate? 5. Is a clear urine always normal? Explain your answer. 2. The urology clinic questions a urinalysis report from the laboratory. The laboratory report states that a reagent strip reading of a specific gravity of 1.020, protein 3 g/dL, and glucose 2 g/dL. The specific gravity in the urology clinic was greater than 1.035. 3920_Ch04_058-070 23/01/14 10:56 AM Page 70 3920_Ch05_071-098 23/01/14 10:13 AM Page 71 CHAPTER 5 Chemical Examination of Urine LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 5-1 Describe the proper technique for performing reagent strip testing. 5-2 List four causes of premature deterioration of reagent strips, and describe how to avoid them. 5-3 List five quality-control procedures routinely performed with reagent strip testing. 5-4 List the reasons for measuring urinary pH, and discuss their clinical applications. 5-5 Discuss the principle of pH testing by reagent strip. 5-6 Differentiate between prerenal, renal, and postrenal proteinuria, and give clinical examples of each. 5-7 Explain the “protein error of indicators,” and list any sources of interference that may occur with this method of protein testing. 5-8 Discuss microalbuminuria including significance, reagent strip tests, and their principles. 5-9 Explain why glucose that is normally reabsorbed in the proximal convoluted tubule may appear in the urine, and state the renal threshold levels for glucose. 5-10 Describe the principle of the glucose oxidase method of reagent strip testing for glucose, and name possible causes of interference with this method. 5-11 Describe the copper reduction method for detection of urinary reducing substances, and discuss the current use of this procedure. 5-14 Differentiate between hematuria, hemoglobinuria, and myoglobinuria with regard to the appearance of urine and serum and clinical significance. 5-15 Describe the chemical principle of the reagent strip method for blood testing, and list possible causes of interference. 5-16 Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen, and finally urobilin. 5-17 Describe the relationship of urinary bilirubin and urobilinogen to the diagnosis of bile duct obstruction, liver disease, and hemolytic disorders. 5-18 Discuss the principle of the reagent strip test for urinary bilirubin, including possible sources of error. 5-19 State two reasons for increased urine urobilinogen and one reason for a decreased urine urobilinogen. 5-20 Discuss the principle of the nitrite-reagent-strip test for bacteriuria. 5-21 List five possible causes of a false-negative result in the reagent strip test for nitrite. 5-22 State the principle of the reagent strip test for leukocytes. 5-23 Discuss the advantages and sources of error of the reagent strip test for leukocytes. 5-24 Explain the principle of the chemical test for specific gravity. 5-12 Name the three “ketone bodies” appearing in urine and three causes of ketonuria. 5-25 Compare reagent strip testing for urine specific gravity with osmolality and refractometer testing. 5-13 Discuss the principle of the sodium nitroprusside reaction to detect ketones, including sensitivity and possible causes of interference. 5-26 Correlate physical and chemical urinalysis results. 3920_Ch05_071-098 23/01/14 10:13 AM Page 72 72 Part Two | Urinalysis KEY TERMS Bacteriuria Hemosiderin Postrenal proteinuria Bilirubin Jaundice Prerenal proteinuria Fanconi syndrome Ketonuria Protein error of indicators Ferritin Leukocyturia Proteinuria Glycosuria Microalbuminuria Stercobilinogen Hematuria Myoglobinuria Urobilinogen Hemoglobinuria Orthostatic proteinuria Uromodulin Reagent Strips Routine chemical examination of urine has changed dramatically since the early days of urine testing, due to the development of the reagent strip method for chemical analysis. Reagent strips currently provide a simple, rapid means for performing medically significant chemical analysis of urine, including pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, and specific gravity. The two major types of reagent strips are manufactured under the trade names Multistix (Siemens Healthcare Diagnostics, Deerfield, IN) and Chemstrip (Roche Diagnostics, Indianapolis, IN). These products are available with single-or multipletesting areas, and the brand and number of tests used are a matter of laboratory preference. Certain variations relating to chemical reactions, sensitivity, specificity, and interfering substances occur among the products and are discussed in the following sections. Reagent strip brands are also specified by instrumentation manufacturers. Reagent strips consist of chemical-impregnated absorbent pads attached to a plastic strip. A color-producing chemical reaction takes place when the absorbent pad comes in contact with urine. The reactions are interpreted by comparing the color produced on the pad within the required time frame with a chart supplied by the manufacturer. Several colors or intensities of a color for each substance being tested appear on the chart. By careful comparison of the colors on the chart and the strip, a semiquantitative value of trace, 1+, 2+, 3+, or 4+ can be reported. An estimate of the milligrams per deciliter present is available for appropriate testing areas. Automated reagent strip readers also provide Système International units. Reagent Strip Technique Testing methodology includes dipping the reagent strip completely, but briefly, into a well-mixed specimen, removing excess urine from the strip by running the edge of the strip on the container when withdrawing it from the specimen, blotting it horizontally on an absorbent medium, waiting the specified length of time for reactions to take place, and comparing the colored reactions against the manufacturer’s chart using a good light source. Errors Caused by Improper Technique 1. Formed elements such as red and white blood cells sink to the bottom of the specimen and will be undetected in an unmixed specimen. 2. Allowing the strip to remain in the urine for an extended period may cause leaching of reagents from the pads. 3. Excess urine remaining on the strip after its removal from the specimen can produce a run-over between chemicals on adjacent pads, producing distortion of the colors. To ensure against run-over, blotting the edge of the strip on absorbent paper and holding the strip horizontally while comparing it with the color chart is recommended. 4. The timing for reactions to take place varies between tests and manufacturers, and ranges from an immediate reaction for pH to 120 seconds for leukocyte esterase. For the best semiquantitative results, the manufacturer’s stated time should be followed; however, when precise timing cannot be achieved, manufacturers recommend that reactions be read between 60 and 120 seconds, with the leukocyte esterase reaction read at 120 seconds. 5. A good light source is essential for accurate interpretation of color reactions. PROCEDURE 5-1 Reagent Strip Technique1,2 1. Dip the reagent strip briefly into a well-mixed uncentrifuged urine specimen at room temperature. 2. Remove excess urine by touching the edge of the strip to the container as the strip is withdrawn. 3. Blot the edge of the strip on a disposable absorbent pad. 4. Wait the specified amount of time for the reaction to occur. 5. Compare the color reaction of the strip pads to the manufacturer’s color chart in good lighting. 3920_Ch05_071-098 23/01/14 10:13 AM Page 73 Chapter 5 | Chemical Examination of Urine 73 6. The strip must be held close to the color chart without actually being placed on the chart. Automated reagent strip instruments standardize the color interpretation and timing of the reaction and are not subject to room lighting deficiencies or inconsistency among laboratory personnel (Appendix A). 7. Reagent strips and color charts from different manufacturers are not interchangeable. 8. Specimens that have been refrigerated must be allowed to return to room temperature prior to reagent strip testing, as the enzymatic reactions on the strips are temperature dependent. Handling and Storing Reagent Strips In addition to using correct testing technique, reagent strips must be protected from deterioration caused by moisture, volatile chemicals, heat, and light. Reagent strips are packaged in opaque containers with a desiccant to protect them from light and moisture. Strips are removed just prior to testing, and the bottle is tightly resealed immediately. Bottles should not be opened in the presence of volatile fumes. Manufacturers recommend that reagent strips be stored at room temperature below 30°C (but never refrigerated). All bottles are stamped with an expiration date that represents the functional life expectancy of the chemical pads. Reagent strips must not be used past the expiration date. Care must be taken not to touch the chemical pads when removing the strips. A visual inspection of the strip should be done each time a strip is used to detect deterioration, even though the strips may still be within the expiration date. Quality Control of Reagent Strips Reagent strips must be checked with both positive and negative controls a minimum of once every 24 hours. Many laboratories perform this check at the beginning of each shift. Testing is also performed when a new bottle of reagent strips is opened, questionable results are obtained, or there is concern about the integrity of the strips. All quality control results must be recorded following laboratory protocol. Several companies manufacture both positive and negative controls. Distilled water is not recommended as a negative control because reagent strip chemical reactions are designed to perform at ionic concentrations similar to urine. All readings of the negative control must be negative, and positive control readings should agree with the published value. Results that do not agree with the published values must be resolved through the testing of additional strips and controls (see Chapter 1). Demonstration of chemically acceptable reagent strips does not entirely rule out the possibility of inaccurate results. Interfering substances in the urine, technical carelessness, and color blindness also produce errors. Reagent strip manufacturers have published information concerning the limitations (e.g., interfering substances, sensitivities) of their chemical reactions, and laboratory personnel should be aware of these conditions. As mentioned in Chapter 4, a primary example of reagent strip interference is the masking of color reactions by the orange pigment present in the urine of persons taking phenazopyridine compounds. If laboratory personnel do not recognize the presence of this pigment or other pigments, they will report many erroneous results. Confirmatory Testing Confirmatory tests are defined as test using different reagents or methodologies to detect the same substances as detected by the reagent strips with the same or greater sensitivity or specificity.4 Nonreagent strip testing procedures using tablets and liquid chemicals may be available when questionable results are obtained or highly pigmented specimens are encountered. In the past, many of these procedures were used routinely to confirm positive results. Increased specificity and sensitivity of reagent strips and the use of automated strip readers have reduced the need for routine use of these procedures.5,6 The chemical reliability of these procedures also must be checked using positive and negative controls. Specific confirmatory tests are discussed in this chapter under their specific sections or the Historical Notes devoted to the chemical parameters for which they are used. The principles and procedures for these tests are included to provide additional information on the principles of the reagent strips and to provide the methodology to perform these tests if necessary. Institutional protocol will determine the situations when they should be performed. pH Along with the lungs, the kidneys are the major regulators of the acid–base content in the body. They do this through the secretion of hydrogen in the form of ammonium ions, hydrogen phosphate, and weak organic acids, and by the reabsorption of bicarbonate from the filtrate in the convoluted tubules (see Chapter 2). A healthy individual usually produces a first morning specimen with a slightly acidic pH of 5.0 to 6.0; a more alkaline pH is found following meals (alkaline tide). The pH of normal random samples can range from 4.5 to 8.0. Consequently, no normal values are assigned to urinary pH, and it must be considered in conjunction with other patient information, such as the acid–base content of the blood, the patient’s renal function, the presence of a urinary tract infection, the patient’s dietary intake, and the age of the specimen (Table 5–1). Clinical Significance The importance of urinary pH is primarily as an aid in determining the existence of systemic acid–base disorders of metabolic or respiratory origin and in the management of urinary conditions that require the urine to be maintained at a specific pH. In respiratory or metabolic acidosis not related to renal function disorders, the urine is acidic; conversely, if respiratory or metabolic alkalosis is present, the 3920_Ch05_071-098 23/01/14 10:14 AM Page 74 74 Part Two | Urinalysis SUMMARY 5-1 Reagent Strip Testing Table 5–1 Causes of Acid and Alkaline Urine Care of Reagent Strips Acid Urine Alkaline Urine 1. Store with desiccant in an opaque, tightly closed container. 2. Store below 30°C; do not freeze. 3. Do not expose to volatile fumes. 4. Do not use past the expiration date. 5. Do not use if chemical pads become discolored. 6. Remove strips immediately prior to use. Technique 1. Mix specimen well. 2. Let refrigerated specimens warm to room temperature before testing. 3. Dip the strip completely, but briefly, into specimen. 4. Remove excess urine by withdrawing the strip against the rim of the container and by blotting the edge of the strip. Emphysema Diabetes mellitus Starvation Dehydration Hyperventilation Vomiting Renal tubular acidosis Presence of ureaseproducing bacteria Vegetarian diet Old specimens 5. Compare reaction colors with the manufacturer’s chart under a good light source at the specified time. 6. Perform backup tests when indicated. 7. Be alert for the presence of interfering substances. 8. Understand the principles and significance of the test; read package inserts. 9. Relate chemical findings to each other and to the physical and microscopic urinalysis results. Quality Control 1. Test open bottles of reagent strips with known positive and negative controls every 24 hours. 2. Resolve control results that are out of range by further testing. 3. Test reagents used in backup tests with positive and negative controls. 4. Perform positive and negative controls on new reagents and newly opened bottles of reagent strips. 5. Record all control results and reagent lot numbers. urine is alkaline. Therefore, a urinary pH that does not conform to this pattern may be used to rule out the suspected condition, or, as discussed in Chapter 3, it may indicate a disorder resulting from the kidneys’ inability to secrete or to reabsorb acid or base. The precipitation of inorganic chemicals dissolved in the urine forms urinary crystals and renal calculi. This precipitation depends on urinary pH and can be controlled by maintaining the urine at a pH that is incompatible with the precipitation of the particular chemicals causing the calculi formation. For example, calcium oxalate, a frequent constituent of renal calculi, precipitates primarily in acidic and not alkaline urine. Therefore, maintaining urine at an alkaline Diarrhea Presence of acid-producing bacteria (Escherichia coli) High-protein diet Cranberry juice Medications (methenamine mandelate [Mandelamine], fosfomycin tromethamine [Monurol]) pH discourages formation of the calculi. Knowledge of urinary pH is important in the identification of crystals observed during microscopic examination of the urine sediment. This will be discussed in detail in Chapter 6. Maintaining an acidic urine can be valuable in treating urinary tract infections caused by urea-splitting organisms because they do not multiply as readily in an acidic medium. These same organisms are also responsible for the highly alkaline pH found in specimens that have been allowed to sit unpreserved for extended periods. Urinary pH is controlled primarily by dietary regulation, although medications also may be used. Persons on high-protein and high-meat diets tend to produce acidic urine, whereas urine from vegetarians is more alkaline, due to the formation of bicarbonate following digestion of many fruits and vegetables. An exception to the rule is cranberry juice, which produces an acidic urine and has long been used as a home remedy for minor bladder infections because it inhibits the colonization of certain urinary pathogens. People who are prone to frequent urinary tract infections are often advised to drink cranberry juice or take over-the-counter cranberry pills. Medications prescribed for urinary tract infections, such as methenamine mandelate (Mandelamine) and fosfomycin tromethamine (Monurol) are metabolized to produce an acidic urine. The pH of freshly excreted urine does not reach above 8.5 in normal or abnormal conditions. A pH above 8.5 is associated with an improperly preserved specimen and indicates that a fresh specimen should be obtained to ensure the validity of the analysis. TECHNICAL TIP Collecting specimens in containers other than the single-use laboratory-supplied containers can produce a pH above 8.5 if alkaline detergent remains in the container. 3920_Ch05_071-098 23/01/14 10:14 AM Page 75 Chapter 5 | Chemical Examination of Urine 75 Clinical Significance of Urine pH SUMMARY 5-2 Respiratory or metabolic acidosis/ketosis Respiratory or metabolic alkalosis Defects in renal tubular secretion and reabsorption of acids and bases—renal tubular acidosis Renal calculi formation and prevention Treatment of urinary tract infections Precipitation/identification of crystals Determination of unsatisfactory specimens Reagent Strip Reactions The Multistix and Chemstrip brands of reagent strips measure urine pH in 0.5- or 1-unit increments between pH 5 and 9. To differentiate pH units throughout this wide range, both manufacturers use a double-indicator system of methyl red and bromthymol blue. Methyl red produces a color change from red to yellow in the pH range 4 to 6, and bromthymol blue turns from yellow to blue in the range of 6 to 9. Therefore, in the pH range 5 to 9 measured by the reagent strips, one sees colors progressing from orange at pH 5 through yellow and green to a final deep blue at pH 9. H+ Methyl red + (Red-orange → H+ → bromthymol blue – yellow) (green → blue) No known substances interfere with urinary pH measurements performed by reagent strips. TECHNICAL TIP Care must be taken to prevent run-over between the pH testing area and the adjacent, highly acidic protein testing area on Multistix, as this may produce a falsely acidic reading in an alkaline urine. SUMMARY 5-3 Reagents Sensitivity Sources of error/ interference: Correlations with other tests: pH Reagent Strip Methyl red, bromthymol blue Multistix: 5.0 to 8.5 in 0.5 increments Chemstrip: 5.0 to 9.0 in 1.0 increments No known interfering substances Run-over from adjacent pads Old specimens Nitrite Leukocytes Microscopic Protein Of the routine chemical tests performed on urine, the most indicative of renal disease is the protein determination. Proteinuria is often associated with early renal disease, making the urinary protein test an important part of any physical examination. Normal urine contains very little protein: usually, less than 10 mg/dL or 100 mg per 24 hours is excreted. This protein consists primarily of low-molecular-weight serum proteins that have been filtered by the glomerulus and proteins produced in the genitourinary tract. Due to its low molecular weight, albumin is the major serum protein found in normal urine. Even though it is present in high concentrations in the plasma, the normal urinary albumin content is low because the majority of albumin presented to the glomerulus is not filtered, and much of the filtered albumin is reabsorbed by the tubules. Other proteins include small amounts of serum and tubular microglobulins; Tamm-Horsfall protein (uromodulin) produced by the renal tubular epithelial cells; and proteins from prostatic, seminal, and vaginal secretions. (Uromodulin is a more recent name for Tamm-Horsfall protein. Uromodulin is routinely produced in the distal convoluted tubule. As will be discussed in Chapter 6, uromodulin forms the matrix of casts formed in the distal convoluted tubule.) Clinical Significance Demonstration of proteinuria in a routine analysis does not always signify renal disease; however, its presence does require additional testing to determine whether the protein represents a normal or a pathologic condition. Clinical proteinuria is indicated at 30 mg/dL or greater (300 mg/L).7 The causes of proteinuria are varied and can be grouped into three major categories: prerenal, renal, and postrenal, based on the origin of the protein. Prerenal Proteinuria As the name implies, prerenal proteinuria is caused by conditions affecting the plasma prior to its reaching the kidney and, therefore, is not indicative of actual renal disease. This condition is frequently transient, caused by increased levels of low-molecular-weight plasma proteins such as hemoglobin, myoglobin, and the acute phase reactants associated with infection and inflammation. The increased filtration of these proteins exceeds the normal reabsorptive capacity of the renal tubules, resulting in an overflow of the proteins into the urine. Because reagent strips detect primarily albumin, prerenal proteinuria is usually not discovered in a routine urinalysis. Bence Jones Protein A primary example of proteinuria due to increased serum protein levels is the excretion of Bence Jones protein by persons with multiple myeloma. In multiple myeloma, a proliferative disorder of the immunoglobulin-producing plasma cells, the serum contains markedly elevated levels of monoclonal immunoglobulin light chains (Bence Jones protein). This lowmolecular-weight protein is filtered in quantities exceeding the tubular reabsorption capacity and is excreted in the urine. 3920_Ch05_071-098 23/01/14 10:14 AM Page 76 76 Part Two | Urinalysis Suspected cases of multiple myeloma must be diagnosed by performing serum electrophoresis and immunoelectrophoresis. The screening test for Bence Jones protein is not routinely performed, as cases of multiple myeloma are easily detected by chemical methods (see the Historical Note, Screening Test for Bence Jones Protein). Renal Proteinuria Proteinuria associated with true renal disease may be the result of either glomerular or tubular damage. Glomerular Proteinuria When the glomerular membrane is damaged, selective filtration is impaired, and increased amounts of serum protein and eventually red and white blood cells pass through the membrane and are excreted in the urine. Conditions that present the glomerular membrane with abnormal substances (e.g., amyloid material, toxic substances, and the immune complexes found in lupus erythematosus and streptococcal glomerulonephritis) are major causes of proteinuria due to glomerular damage. Increased pressure from the blood entering the glomerulus may override the selective filtration of the glomerulus, causing increased albumin to enter the filtrate. This condition may be reversible, such as occurs during strenuous exercise and dehydration or is associated with hypertension. Proteinuria that occurs during the latter months of pregnancy may indicate a pre-eclamptic state and should be considered by the physician in conjunction with other clinical symptoms, such as hypertension, to determine if this condition exists. The discovery of protein, particularly in a random sample, is not always of pathologic significance, because several benign causes of renal proteinuria exist. Benign proteinuria is usually transient and can be produced by conditions such as strenuous exercise, high fever, dehydration, and exposure to cold. Microalbuminuria The development of diabetic nephropathy leading to reduced glomerular filtration and eventual renal failure is a common HISTORICAL NOTE Screening Test for Bence Jones Protein Unlike other proteins, which coagulate and remain coagulated when exposed to heat, Bence Jones protein coagulates at temperatures between 40°C and 60°C and dissolves when the temperature reaches 100°C. Therefore, a specimen that appears turbid between 40°C and 60°C and clear at 100°C can be suspected of containing Bence Jones protein. Interference due to other precipitated proteins can be removed by filtering the specimen at 100°C and observing the specimen for turbidity as it cools to between 40°C and 60°C. occurrence in persons with both type 1 and type 2 diabetes mellitus. Onset of renal complications can first be predicted by detection of microalbuminuria, and the progression of renal disease can be prevented through better stabilization of blood glucose levels and control of hypertension. The presence of microalbuminuria is also associated with an increased risk of cardiovascular disease. 8, 9 Orthostatic (Postural) Proteinuria A persistent benign proteinuria occurs frequently in young adults and is termed orthostatic proteinuria, or postural proteinuria. It occurs following periods spent in a vertical posture and disappears when a horizontal position is assumed. Increased pressure on the renal vein when in the vertical position is believed to account for this condition. Patients suspected of orthostatic proteinuria are requested to empty the bladder before going to bed, collect a specimen immediately upon arising in the morning, and collect a second specimen after remaining in a vertical position for several hours. Both specimens are tested for protein, and if orthostatic proteinuria is present, a negative reading will be seen on the first morning specimen, and a positive result will be found on the second specimen. Tubular Proteinuria Increased albumin is also present in disorders affecting tubular reabsorption because the normally filtered albumin can no longer be reabsorbed. Other low-molecular-weight proteins that are usually reabsorbed are also present. Causes of tubular dysfunction include exposure to toxic substances and heavy metals, severe viral infections, and Fanconi syndrome. The amount of protein that appears in the urine following glomerular damage ranges from slightly above normal to 4 g/day, whereas markedly elevated protein levels are seldom seen in tubular disorders. Postrenal Proteinuria Protein can be added to a urine specimen as it passes through the structures of the lower urinary tract (ureters, bladder, urethra, prostate, and vagina). Bacterial and fungal infections HISTORICAL NOTE Microalbuminuria Testing Before the development of current reagent strip methods that are specific for albumin, detection of microalbuminuria required collection of a 24-hour urine specimen. Specimens were tested using quantitative procedures for albumin. Results were reported in mg of albumin/24 hours or as the albumin excretion (AER) in µg/min. With these methods, microalbumin was considered significant when 30 to 300 mg of albumin is excreted in 24 hours or the AER is 20 to 200 µg/min. 3920_Ch05_071-098 23/01/14 10:14 AM Page 77 Chapter 5 | Chemical Examination of Urine 77 and inflammations produce exudates containing protein from the interstitial fluid. The presence of blood as the result of injury or menstrual contamination contributes protein, as does the presence of prostatic fluid and large amounts of spermatozoa. Reagent Strip Reactions Traditional reagent strip testing for protein uses the principle of the protein error of indicators to produce a visible colorimetric reaction. Contrary to the general belief that indicators produce specific colors in response to particular pH levels, certain indicators change color in the presence of protein even though the pH of the medium remains constant. This is because protein (primarily albumin) accepts hydrogen ions from the indicator. The test is more sensitive to albumin because albumin contains more amino groups to accept the hydrogen ions than other proteins. Depending on the manufacturer, the protein area of the strip contains either tetrabromophenol blue (Multistix) or 3',3",5',5"-tetrachlorophenol, 3,4,5,6-tetrabromosulfonphthalein (Chemstrip), and an acid buffer to maintain the pH at a constant level. At a pH level of 3, both indicators appear yellow in the absence of protein; however, as the protein concentration increases, the color progresses through various shades of green and finally to blue. Readings are reported in terms of negative, trace, 1+, 2+, 3+, and 4+; or the semiquantitative values of 30, 100, 300, or 2000 mg/dL corresponding to each color change. Trace values are considered to be less than 30 mg/dL. Interpretation of trace readings SUMMARY 5-4 Clinical Significance of Urine Protein Prerenal Intravascular hemolysis Muscle injury Acute phase reactants Multiple myeloma Tubular Disorders Fanconi syndrome Toxic agents/heavy metals Severe viral infections Renal Glomerular disorders Postrenal Lower urinary tract infections/ inflammation Injury/trauma Immune complex disorders Amyloidosis Menstrual contamination Toxic agents Prostatic fluid/spermatozoa Diabetic nephropathy Vaginal secretions Strenuous exercise Dehydration Hypertension Pre-eclampsia Orthostatic or postural proteinuria can be difficult. Reporting of trace values may be a laboratory option. Indicator + protein (Yellow) pH 3.0 protein + H+ indicator – H+ (blue-green) Reaction Interference The major source of error with reagent strips occurs with highly buffered alkaline urine that overrides the acid buffer system, producing a rise in pH and a color change unrelated to protein concentration. Likewise, a technical error of allowing the reagent pad to remain in contact with the urine for a prolonged period may remove the buffer. False-positive readings are obtained when the reaction does not take place under acidic conditions. Highly pigmented urine and contamination of the container with quaternary ammonium compounds, detergents, and antiseptics also cause false-positive readings. A false-positive trace reading may occur in specimens with a high specific gravity. Sulfosalicylic Acid Precipitation Test The sulfosalicylic acid (SSA) test is a cold precipitation test that reacts equally with all forms of protein. Various concentrations and amounts of SSA can be used to precipitate protein, and methods vary greatly among laboratories. All precipitation tests must be performed on centrifuged specimens to remove any extraneous contamination. Based on the protocol of the laboratory, an SSA test may be performed in certain situations. The procedure is included in this section to serve as a reference if needed (Procedure 5–2).5 Testing for Microalbuminuria Several semiquantitative reagent strip methods have been developed so that patients at risk for renal disease can be monitored using random or first morning specimens. These methods are based on immunochemical assays for albumin or albumin-specific reagent strips that also measure creatinine to produce an albumin:creatinine ratio. Immunochemical assays include the Micral-Test (Roche Diagnostics, Indianapolis, IN) and the ImmunoDip (Sakisui Diagnostics, Framingham, MA). Both reagent strips are read visually, and first morning specimens are recommended. Micral-Test reagent strips contain a gold-labeled antihuman albumin antibody-enzyme conjugate. Strips are dipped into the urine up to a level marked on the strip and held for 5 seconds. Albumin in the urine binds to the antibody. The bound and unbound conjugates move up the strip by wicking TECHNICAL TIP The specific gravity of the urine specimen should be considered in evaluating urine protein because a trace protein in a dilute specimen is more significant than in a concentrated specimen. 3920_Ch05_071-098 23/01/14 10:14 AM Page 78 78 Part Two | Urinalysis SUMMARY 5-5 Protein Reagent Strip Reagents Multistix: Tetrabromophenol blue Chemstrip: 3’,3’’,5’,5’’-tetrachlorophenol 3,4,5,6-tetrabromosulfophthalein Sensitivity Multistix: 15 to 30 mg/dL albumin Chemstrip: 6 mg/dL albumin Sources of error/interference False-positive: Highly buffered interference alkaline urine Pigmented specimens, phenazopyridine Quaternary ammonium compounds (detergents) Antiseptics, chlorhexidine Loss of buffer from prolonged exposure of the strip to the specimen reagent High specific gravity False-negative Proteins other than albumin Microalbuminuria Correlations Blood with other tests: Nitrite Leukocytes Microscopic action. Unbound conjugates are removed in a captive zone by combining with albumin embedded in the strip. The urine albumin–bound conjugates continue up the strip and reach an area containing enzyme substrate. The conjugated enzyme reacts with the substrate, producing colors ranging from white to red. The amount of color produced represents the amount of albumin present in the urine. The color is compared with a chart on the reagent strip bottle after 1 minute. Results range from 0 to 10 mg/dL. The ImmunoDip reagent strip uses an immunochromographic technique. Strips are individually packaged in specially designed containers. The container is placed in the urine specimen for 3 minutes. A controlled amount of urine enters the container through a vent hole. The urine encounters blue latex particles coated with antihuman albumin antibody. Albumin in the urine binds with the coated particles. The bound and unbound particles continue to migrate up the strip. The migration is controlled by the size of the particles; unbound particles do not migrate as far as the bound particles. First a blue band is formed by the unbound particles. The bound particles continue to migrate and form a second blue band further up the strip. The top band therefore represents the bound particles (urine albumin) and the bottom band represents unbound PROCEDURE 5-2 Sulfosalicylic Acid Precipitation Test 1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine. 2. Mix by inversion and observe for cloudiness. 3. Grade the degree of turbidity (see table, following). Table Reporting SSA Turbidity Grade Turbidity Negative No increase in turbidity Noticeable turbidity Distinct turbidity, no granulation Turbidity, granulation, no flocculation Turbidity, granulation, flocculation Clumps of protein Trace 1+ 2+ 3+ 4+ Protein Range (mg/dL) Less than 6 6–30 30–100 100–200 200–400 Greater than 400 particles. The color intensity of the bands is compared against the manufacturer’s color chart. A darker bottom band represents less than 1.2 mg/dL, equal band colors represent 1.2 to 1.8 mg/dL, and a darker top band represents 2.0 to 8.0 mg/dL of albumin. A darker bottom band is negative, equal band color is borderline, and a darker top band represents positive results. Albumin:Creatinine Ratio The Clinitek Microalbumin reagent strips and the Multistix Pro reagent strips (Siemens Healthcare Diagnostics, Deerfield, IN) provide simultaneous measurement of albumin/protein and creatinine that permits an estimation of the 24-hour microalbumin excretion.10 As discussed in Chapter 3, creatinine is produced and excreted at a consistent rate for each individual. Therefore, by comparing the albumin excretion to the creatinine excretion, the albumin reading can be corrected for overhydration and dehydration in a random sample. In addition to including creatinine on the reagent strip, the albumin low-test pad is changed to a dye-binding reaction that is more specific for albumin than the protein error of indicators’ reaction on strips measuring protein. Reagent Strip Reactions Albumin Albumin reagent strips use the dye bis(3',3"-diiodo-4', 4"-dihydroxy-5',5"-dinitrophenyl)-3,4,5,6-tetrabromo sulphonphthalein (DIDNTB), which has a higher sensitivity 3920_Ch05_071-098 23/01/14 10:14 AM Page 79 Chapter 5 | Chemical Examination of Urine 79 and specificity for albumin. Whereas conventional protein reagent pads have a sensitivity of 30 mg/dL or greater and may include proteins other than albumin, the DIDNTB strips can measure albumin between 8 and 15 mg/dL (80 to 150 mg/L) without inclusion of other proteins. Reaction interference by highly buffered alkaline urine (always a concern with conventional reagent strips) is controlled by using paper treated with bis-(heptapropylene glycol) carbonate. Addition of polymethyl vinyl ether decreases the nonspecific binding of polyamino acids to the albumin pad. Colors range from pale green to aqua blue. Falsely elevated results can be caused by visibly bloody urine, and abnormally colored urines may interfere with the readings.2 Creatinine The principle of the reagent strip for creatinine is based on the pseudoperoxidase activity of copper-creatinine complexes. The reaction follows the same principle as the reaction for blood on the reagent strips discussed later in this chapter. Reagent strips contain copper sulfate (CuSO4), 3,3',5,5'-tetramethylbenzidine (TMB), and diisopropyl benzene dihydroperoxide (DBDH). Creatinine in the urine combines with the copper sulfate to form copper-creatinine peroxidase. This reacts with the peroxide DBDH, releasing oxygen ions that oxidize the chromogen TMB and producing a color change from orange through green to blue.7 CuSO4 + CRE → Cu(CRE) peroxidase Cu(CRE) peroxidase DBDH + TMB (peroxidase) (chromogen) oxidized TMB + H2O (orange to blue) Results are reported as 10, 50, 100, 200, 300 mg/dL, or 0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine. Reagent strips are unable to detect the absence of creatinine. Falsely elevated results can be caused by visibly bloody urine and the presence of the gastric acid–reducing medication cimetidine (Tagamet). Abnormally colored urines also may interfere with the readings. No creatinine readings are considered abnormal, as creatinine is normally present in concentrations of 10 to 300 mg/dL. The purpose of the creatinine measurement is to correlate the albumin concentration to the urine concentration, producing a semiquantitative albumin:creatinine ratio (A:C) ratio. Albumin/Protein:Creatinine Ratio Automated and manual methods are available for determining the A:C ratio based on the previously discussed reactions. The Clinitek Microalbumin reagent strips are designed for instrumental use only. Strips are read on Clinitek Urine Chemistry Analyzers. The strips measure only albumin and creatinine and calculate the A:C ratio. Results are displayed and printed out for albumin, creatinine, and the A:C ratio in both conventional and S.I. units. Abnormal results for the A:C ratio are 30 to 300 mg/g or 3.4 to 33.9 mg/mmol.10 The Siemens Multistix Pro 10 reagent strips include reagent pads for creatinine, protein-high and protein-low (albumin), along with pads for glucose, ketones, blood, nitrite, leukocyte esterase, pH, bilirubin, and specific gravity. Urobilinogen is not included on these strips. The strips can be read manually or on automated Clinitek instruments. The protein-high reaction uses the protein error of indicators principle and the protein-low reaction is the previously discussed dye-binding method. Results are reported as the protein:creatinine ratio, although the protein-low result is used in the calculation. Results from the Clinitek are automatically calculated. Results are reported as normal or abnormal. A result of normal dilute indicates that the specimen should be recollected, making sure it is a first morning specimen. When the reagent strip is read manually, a manufacturersupplied chart is used to determine the ratio based on the results of the protein-high, protein-low, and creatinine readings. When using this chart, the higher of the protein-low or protein-high result is used2 (Fig. 5–1). Glucose Because of its value in the detection and monitoring of diabetes mellitus, the glucose test is the most frequently performed chemical analysis on urine. Due to the nonspecific symptoms associated with the onset of diabetes, it is estimated that more than half of the cases in the world are undiagnosed. Therefore, blood and urine glucose tests are included in all physical examinations and are often the focus of mass health screening programs. Early diagnosis of diabetes mellitus through blood and urine glucose tests provides a greatly improved prognosis. Using currently available reagent strip methods for both blood and urine glucose testing, patients can monitor themselves at home and can detect regulatory problems prior to the development of serious complications. Clinical Significance Under normal circumstances, almost all the glucose filtered by the glomerulus is actively reabsorbed in the proximal convoluted tubule; therefore, urine contains only minute amounts of glucose. Tubular reabsorption of glucose is by active transport in response to the body’s need to maintain an adequate concentration of glucose. Should the blood level of glucose become elevated (hyperglycemia), as occurs in diabetes mellitus, Reported Protein Result (mg/dL) 10 Negative Recollect* 15 30 100, 300, or 2000 Creatinine Result (mg/dL) 50 100 200 No 300 Ab rm no al rm al *Specimen is too dilute to determine ratio result accurately. Repeat test on new specimen, preferably a first-morning collection. Figure 5–1 A protein:creatinine ratio determination chart. (Image adapted from Bayer HealthCare LLC, Elkhart, IN.) 3920_Ch05_071-098 23/01/14 10:14 AM Page 80 80 Part Two | Urinalysis SUMMARY 5-6 Microalbumin Testing Immunologic Tests Micral-Test Principle: Enzyme immunoassay Sensitivity: 0 to 10 mg/dL Reagents: Gold-labeled antibody B-galactosidase Chlorophenol red galactoside Interference: False-negative: Dilute urine ImmunoDip Principle: Immunochromographics Sensitivity: 1.2 to 8.0 mg/dL Reagents: Antibody-coated blue latex particles Interference: False-negative: Dilute urine Albumin:Creatinine Ratio Clinitest Microalbumin Strips/Multistix-Pro Principle: Sensitive albumin tests related to creatinine concentration to correct for patient hydration Reagents: Albumin: dye bis(3’,3’’-diiodo-4’,4’’-dihydroxy-5’,5’’dinitrophenyl)-3,4,5,6-tetrabromo sulphonphtalein (DIDNTB) Creatinine: copper sulfate (CuSO4), 3,3’,5,5’-tetramethylbenzidine (TMB), and diisopropyl benzene dihydroperoxide (DBDH) Sensitivity: Albumin: 10 to 150 mg/L Creatinine: 10 to 300 mg/dL, 0.9 to 26.5 mmol/L Interference: Visibly bloody or abnormally colored urine Creatinine: Cimetidine: False positive Hyperglycemia that occurs during pregnancy and disappears after delivery is called gestational diabetes. The onset of the hyperglycemia and glycosuria is normally around the sixth month of pregnancy, although glycosuria may occur sooner. Hormones secreted by the placenta block the action of insulin, resulting in insulin resistance and hyperglycemia. Detection of gestational diabetes is important to the welfare of the baby, because glucose crosses the placenta whereas insulin does not. The baby develops high glucose levels, causing the baby’s pancreas to produce more insulin. The excess glucose presented to the baby is stored as fat, resulting in a large baby (macrosomia) at risk for obesity and later type 2 diabetes. Women who have gestational diabetes also are prone to developing type 2 diabetes mellitus in later years. Hyperglycemia of nondiabetic origin is seen in a variety of disorders and also produces glycosuria. Many of these disorders are associated with hormonal function and include pancreatitis, acromegaly, Cushing syndrome, hyperthyroidism, pheochromocytoma, and thyrotoxicosis. The hormones glucagon, epinephrine, cortisol, thyroxine, and growth hormone, which are increased in these disorders, work in opposition to insulin, thereby producing hyperglycemia and glucosuria. Whereas a primary function of insulin is to convert glucose to glycogen for storage (glycogenesis), these opposing hormones cause the breakdown of glycogen to glucose (glycogenolysis), resulting in increased levels of circulating glucose. Epinephrine is also a strong inhibitor of insulin secretion and is increased when the body is subjected to severe stress, which accounts for the glucosuria seen in conjunction with cerebrovascular trauma and myocardial infarction. Glycosuria occurs in the absence of hyperglycemia when the reabsorption of glucose by the renal tubules is compromised. This is frequently referred to as “renal glycosuria” and is seen in end-stage renal disease, cystinosis, and Fanconi syndrome. Glycosuria not associated with gestational diabetes is occasionally seen as a result of a temporary lowering of the renal threshold for glucose during pregnancy. SUMMARY 5-7 the tubular transport of glucose has reached its renal threshold, and glucose appears in the urine. The blood level at which tubular reabsorption stops (renal threshold) for glucose is approximately 160 to 180 mg/dL. Blood glucose levels fluctuate, and a nonfasting normal person may have glycosuria following a meal containing a high glucose content. Therefore, the most informative glucose results are obtained from specimens collected under controlled conditions. Fasting prior to the collection of samples for screening tests is recommended. For purposes of diabetes monitoring, specimens are usually tested 2 hours after meals. A first morning specimen does not always represent a fasting specimen because glucose from an evening meal may remain in the bladder overnight, and patients should be advised to empty the bladder and collect the second specimen.2 Clinical Significance of Urine Glucose Hyperglycemia-Associated Renal-Associated Diabetes mellitus Pancreatitis Pancreatic cancer Acromegaly Cushing syndrome Hyperthyroidism Pheochromocytoma Central nervous system damage Stress Gestational diabetes Fanconi syndrome Advanced renal disease Osteomalacia Pregnancy 3920_Ch05_071-098 23/01/14 10:14 AM Page 81 Chapter 5 | Chemical Examination of Urine 81 Reagent Strip (Glucose Oxidase) Reaction The glucose oxidase procedure provides a specific test for glucose. Reagent strips employ the glucose oxidase testing method by impregnating the testing area with a mixture of glucose oxidase, peroxidase, chromogen, and buffer to produce a double sequential enzyme reaction. In the first step, glucose oxidase catalyzes a reaction between glucose and room air (oxygen) to produce gluconic acid and peroxide. In the second step, peroxidase catalyzes the reaction between peroxide and chromogen to form an oxidized colored compound that is produced in direct proportion to the concentration of glucose. 1. Glucose + O2 (air) 2. H2O2 + chromogen SUMMARY 5-8 Reagents Sensitivity Glucose oxidase gluconic acid + H2O2 Peroxidase oxidized colored chromogen + H2O Reagent strip manufacturers use several different chromogens, including potassium iodide (green to brown) (Multistix) and tetramethylbenzidine (yellow to green) (Chemstrip). Urine glucose may be reported in terms of negative, trace, 1+, 2+, 3+, and 4+; however, the color charts also provide quantitative measurements ranging from 100 mg/dL to 2 g/dL, or 0.1% to 2%. The American Diabetes Association recommends quantitative reporting. Reaction Interference Because the glucose oxidase method is specific for glucose, false-positive reactions are not obtained from other urinary constituents, including reducing sugars that may be present. Falsepositive reactions may occur, however, if containers become contaminated with peroxide or strong oxidizing detergents. Substances that interfere with the enzymatic reaction or strong reducing agents, such as ascorbic acid, that prevent oxidation of the chromogen may produce false-negative results. To minimize interference from ascorbic acid, reagent strip manufacturers are incorporating additional chemicals into the test pads. An example is iodate that oxidizes ascorbic acid so that it cannot interfere with the oxidation of the chromogen. Product literature should be carefully reviewed for current information regarding all interfering substances. High levels of ketones also affect glucose oxidase tests at low glucose concentrations; however, because high levels of ketones are usually accompanied by marked glycosuria, this seldom presents a problem. High specific gravity and low temperature may decrease the sensitivity of the test. By far the greatest source of false-negative glucose results is the technical error of allowing specimens to remain unpreserved at room temperature for extended periods, subjecting the glucose to bacterial degradation. Copper Reduction Test (Clinitest) Measurement of glucose by the copper reduction method was one of the earliest chemical tests performed on urine. The test relies on the ability of glucose and other substances to reduce Interference Correlations with other tests Glucose Reagent Strip Multistix Glucose oxidase Peroxidase Potassium iodide Chemstrip Glucose oxidase Peroxidase Tetramethylbenzidine Multistix: 75 to 125 mg/dL Chemstrip: 40 mg/dL False-positive Contamination by oxidizing agents and detergents False-negative High levels of ascorbic acid High levels of ketones High specific gravity Low temperatures Improperly preserved specimens Ketones Protein copper sulfate to cuprous oxide in the presence of alkali and heat. A color change progressing from a negative blue (CuSO4) through green, yellow, and orange/red (Cu2O) occurs when the reaction takes place. CuSO4 (cupric sulfide) + reducing substance Heat Alkali Cu2O (cuprous oxide) + oxidized substance → color (blue/green → orange/red) The classic Benedict solution was developed in 1908 and contained copper sulfate, sodium carbonate, and sodium citrate buffer.11 Urine was added to the solution, heat was applied, and the resulting precipitate was observed for color. A more convenient method that employs Benedict’s principle is the Clinitest tablet (Siemens Healthcare Diagnostics, Deerfield, IN). The tablets contain copper sulfate, sodium carbonate, sodium citrate, and sodium hydroxide. Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of sodium hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium carbonate to prevent room air from interfering with the reduction reaction. Thick-walled tubes should be placed in a heatresistant rack and not held in the hand because the reaction heat could cause a burn. At the conclusion of the effervescent 3920_Ch05_071-098 23/01/14 10:14 AM Page 82 82 Part Two | Urinalysis reaction, the tube is gently shaken, and the color ranging from blue to orange/red can be compared with the manufacturer’s color chart to determine the approximate amount of reducing substance. Care must be taken to observe the reaction closely as it is taking place, because at high glucose levels, a phenomenon known as “pass through” may occur. When this happens, the color produced passes through the orange/red stage and returns to a green-brown color, and if not observed, a high glucose level may be reported as negative. An alternate method using two drops instead of five drops of urine can minimize the occurrence of “pass through.” A separate color chart must be used to interpret the reaction. This chart provides values up to 5 g/dL, whereas the five-drop method is limited to 2 g/dL. The sensitivity of Clinitest to glucose is reduced to a minimum of 200 mg/dL so the Clinitest cannot be used as a confirmatory test for glucose. As a nonspecific test for reducing substances, Clinitest is subject to interference from other reducing sugars, including galactose, lactose, fructose, maltose, pentoses, ascorbic acid, certain drug metabolites, and antibiotics such as the cephalosporins. Therefore, Clinitest does not provide a confirmatory test for glucose. Clinitest tablets are very hygroscopic and should be stored in their tightly closed packages. A strong blue color in the unused tablets suggests deterioration due to moisture accumulation, as does vigorous tablet fizzing. Clinical Significance of Clinitest In addition to glucose, commonly found reducing sugars include galactose, fructose, pentose, and lactose, of which galactose is the most clinically significant. Galactose in the urine of a newborn represents an “inborn error of metabolism” in which lack of the enzyme galactose-1-phosphate uridyl transferase PROCEDURE 5-3 Clinitest Procedure 1. Place a thick glass test tube in a rack; add 5 drops of urine. 2. Add 10 drops of distilled water to the urine in the test tube. 3. Drop one Clinitest tablet into the test tube and observe the reaction until completion (cessation of boiling). CAUTION: The reaction mixture gets very hot. Do not touch the bottom area of the test tube. Use thick glass test tube only. 4. Wait 15 seconds after boiling has stopped and gently shake the contents of the tube. 5. Compare the color of the mixture to the Clinitest color chart and record the result in mg/dL or percent. Observe for the possibility of the “pass-through” phenomenon. If present, repeat the procedure using 2 drops of urine instead of 5 drops. prevents breakdown of ingested galactose and results in failure to thrive and other complications, including death. All states have incorporated screening for galactosemia into their required newborn screening programs (see Chapter 8) because early detection followed by dietary restriction can control the condition. Depending on the laboratory population Clinitest is often performed on pediatric specimens from patients up to at least the age of 2 years. The appearance of other reducing sugars is usually of minimal clinical significance, and lactose is frequently found in the urine of nursing mothers. Ketones The term “ketones” represents three intermediate products of fat metabolism, namely, acetone (2%), acetoacetic acid (20%), and β -hydroxybutyrate (78%). Normally, measurable amounts of ketones do not appear in the urine, because all the metabolized fat is completely broken down into carbon dioxide and water. However, when the use of available carbohydrate as the major source of energy becomes compromised, body stores of fat must be metabolized to supply energy. Ketones are then detected in urine. Clinical Significance Clinical reasons for increased fat metabolism include the inability to metabolize carbohydrate, as occurs in diabetes mellitus; increased loss of carbohydrate from vomiting; and inadequate intake of carbohydrate associated with starvation and malabsorption. Testing for urinary ketones is most valuable in the management and monitoring of insulin-dependent (type 1) diabetes mellitus. Ketonuria shows a deficiency in insulin, indicating the need to regulate dosage. It is often an early indicator of insufficient insulin dosage in type 1 diabetes and in patients with diabetes who experience medical problems in addition to diabetes. Increased accumulation of ketones in the blood leads to electrolyte imbalance, dehydration, and, if not corrected, acidosis and eventual diabetic coma. The use of multiple-test strips in hospital laboratories often produces positive ketone tests unrelated to diabetes because the patient’s illness either prevents adequate intake or absorption of carbohydrates or produces an accelerated loss, as in the case of vomiting. Weight-loss and eating disorder clinics can use a practical application of ketonuria produced by avoidance of carbohydrates to monitor patients. Frequent strenuous exercise can cause overuse of available carbohydrates and produce ketonuria. TECHNICAL TIP Keep in mind that table sugar is sucrose, a nonreducing sugar, and does not react with Clinitest or glucose oxidase strips and therefore cannot be used as a control or in preparation of a laboratory exercise for glucose testing. 3920_Ch05_071-098 23/01/14 10:14 AM Page 83 Chapter 5 | Chemical Examination of Urine 83 SUMMARY 5-9 Clinical Significance of Urine Ketones Diabetic acidosis Insulin dosage monitoring Starvation Malabsorption/pancreatic disorders Strenuous exercise Vomiting Inborn errors of amino acid metabolism (see Chapter 8) SUMMARY 5-10 Ketone Reagent Strip Reagents: Sodium nitroprusside Glycine (Chemstrip) Multistix: 5 to 10 mg/dL acetoacetic acid Chemstrip: 9 mg/dL acetoacetic acid; 70 mg/dL acetone False-positive: Phthalein dyes Highly pigmented red urine Levodopa Medications containing free sulfhydryl groups False-negative: Improperly preserved specimens Glucose Sensitivity: Interference: Reagent Strip Reactions The three ketone compounds are not present in equal amounts in urine. Both acetone and β -hydroxybutyric acid are produced from acetoacetic acid (Fig. 5–2). The proportions of 78% β -hydroxybutyric acid, 20% acetoacetic acid, and 2% acetone are relatively constant in all specimens. Reagent strip tests use the sodium nitroprusside (nitroferricyanide) reaction to measure ketones. In this reaction, acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color. The test does not measure β -hydroxybutyrate and is only slightly sensitive to acetone when glycine is also present; however, inasmuch as these compounds are derived from acetoacetic acid, their presence can be assumed, and it is not necessary to perform individual tests. Results are reported qualitatively as negative, trace, small (1+), moderate (2+), or large (3+), or semiquantitatively as negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large (80 to 160 mg/dL). Acetoacetate (and acetone) + sodium nitroprusside + (glycine) alkaline purple color Reaction Interference Large amounts of levodopa and medications containing sulfhydryl groups, including mercaptoethane sulfonate sodium (MESNA) and captopril, may produce atypical color reactions. Reactions with interfering substances frequently fade on standing, whereas color development from acetoacetic acid increases, resulting in false-positive results from improperly timed readings. Falsely decreased values due to the volatilization of acetone and the breakdown of acetoacetic acid by bacteria are seen in improperly preserved specimens. The Acetest tablet test has been used as a confirmatory test for questionable reagent strip results; however, it was primarily used for testing serum and other bodily fluids and dilutions of these fluids for severe ketosis. Currently, new methods Correlations: measuring β -hydroxybutyrate using reagent strips have been developed to provide automated methods for testing serum and other body fluids. Notice in Figure 5–2 the ketone with the highest concentration is β -hydroxybutyrate. Acetest Tablets Acetest (Siemens Healthcare Diagnostics Inc., Deerfield, IL) provides sodium nitroprusside, glycine, disodium phosphate, and lactose in tablet form. The addition of lactose gives better color differentiation. Acetest tablets are hygroscopic; if the specimen is not completely absorbed within 30 seconds, a new tablet should be used. See Procedure 5–4. Blood Blood may be present in the urine either in the form of intact red blood cells (hematuria) or as the product of red blood cell destruction, hemoglobin (hemoglobinuria). As discussed in Chapter 2, blood present in large quantities can be detected visually; hematuria produces a cloudy red urine, and hemoglobinuria appears as a clear red specimen. Because any amount of blood greater than five cells per microliter of urine is considered clinically significant, visual examination cannot be relied upon to detect the presence of blood. Microscopic examination of the urinary sediment shows intact red blood cells, but free hemoglobin produced either by hemolytic OH O O +2H CH3 C CH2 COOH CH3 C CH2 COOH –CO2 CH3 C –2H Figure 5–2 Production of acetone and butyrate from acetoacetic acid. H -Hydroxybutyrate Acetoacetic acid Acetone CH3 3920_Ch05_071-098 23/01/14 10:15 AM Page 84 84 Part Two | Urinalysis PROCEDURE 5-4 Acetest Procedure 1. Remove the Acetest tablet from the bottle and place on a clean, dry piece of white paper. 2. Place 1 drop of urine on top of the tablet. 3. Wait 30 seconds. 4. Compare the tablet color with the manufacturersupplied color chart. 5. Report as negative, small, moderate, or large. disorders or lysis of red blood cells is not detected. Therefore, chemical tests for hemoglobin provide the most accurate means for determining the presence of blood. Once blood has been detected, the microscopic examination can be used to differentiate between hematuria and hemoglobinuria. Clinical Significance The finding of a positive reagent strip test result for blood indicates the presence of red blood cells, hemoglobin, or myoglobin. Each of these has a different clinical significance. Hematuria Hematuria is most closely related to disorders of renal or genitourinary origin in which bleeding is the result of trauma or damage to the organs of these systems. Major causes of hematuria include renal calculi, glomerular diseases, tumors, trauma, pyelonephritis, exposure to toxic chemicals, and anticoagulant therapy. The laboratory is frequently requested to perform a urinalysis when patients presenting with severe back and abdominal pain are suspected of having renal calculi. In such cases, hematuria is usually of a small to moderate degree, but its presence can be essential to the diagnosis. Hematuria of nonpathologic significance is observed following strenuous exercise and during menstruation. Hemoglobinuria Hemoglobinuria may result from the lysis of red blood cells produced in the urinary tract, particularly in dilute, alkaline urine. It also may result from intravascular hemolysis and the subsequent filtering of hemoglobin through the glomerulus. Lysis of red blood cells in the urine usually shows a mixture of hemoglobinuria and hematuria, whereas no red blood cells are seen in cases of intravascular hemolysis. Under normal conditions, the formation of large hemoglobin-haptoglobin complexes in the circulation prevents the glomerular filtration of hemoglobin. When the amount of free hemoglobin present exceeds the haptoglobin content—as occurs in hemolytic anemias, transfusion reactions, severe burns, brown recluse spider bites, infections, and strenuous exercise— hemoglobin is available for glomerular filtration. Reabsorption of filtered hemoglobin also results in the appearance of large yellow-brown granules of denatured ferritin called hemosiderin in the renal tubular epithelial cells and in the urine sediment. Myoglobinuria Myoglobin, a heme-containing protein found in muscle tissue, not only reacts positively with the reagent strip test for blood but also produces a clear red-brown urine. In myoglobinuria, the presence of myoglobin rather than hemoglobin should be suspected in patients with conditions associated with muscle destruction (rhabdomyolysis). Examples of these conditions include trauma, crush syndromes, prolonged coma, convulsions, muscle-wasting diseases, alcoholism, heroin abuse, and extensive exertion. The development of rhabdomyolysis has been found to be a side effect in certain patients taking the cholesterol-lowering statin medications.12 The heme portion of myoglobin is toxic to the renal tubules, and high concentrations can cause acute renal failure. The massive hemoglobinuria seen in hemolytic transfusion reactions also is associated with acute renal failure. Reagent Strip Reactions Chemical tests for blood use the pseudoperoxidase activity of hemoglobin to catalyze a reaction between the heme component of both hemoglobin and myoglobin and the chromogen tetramethylbenzidine to produce an oxidized chromogen, which has a green-blue color. H2O2 + chromogen Hemoglobin Peroxidase oxidized chromogen + H2O SUMMARY 5-11 Clinical Significance of a Positive Reaction for Blood Hematuria Renal calculi Glomerulonephritis Pyelonephritis Tumors Trauma Exposure to toxic chemicals Anticoagulants Strenuous exercise Hemoglobinuria Transfusion reactions Hemolytic anemias Severe burns Infections/malaria Strenuous exercise/red blood cell trauma Brown recluse spider bites Myoglobinuria Muscular trauma/crush syndromes Prolonged coma Convulsions Muscle-wasting diseases Alcoholism/overdose Drug abuse Extensive exertion Cholesterol-lowering statin medications 3920_Ch05_071-098 23/01/14 10:15 AM Page 85 Chapter 5 | Chemical Examination of Urine 85 HISTORICAL NOTE SUMMARY 5-12 Hemoglobinuria Versus Myoglobinuria Reagents Prior to the development of sensitive serum immunoassay tests for myoglobin, a procedure was used to differentiate between hemoglobin and myoglobin in the urine. A precipitation test was used to screen for the presence of myoglobin; 2.8 g of ammonium sulfate are added to 5 mL of centrifuged urine. After mixing and allowing the specimen to sit for 5 minutes, the urine is filtered or centrifuged, and the supernatant is tested for a reaction for blood with a reagent strip. The principle of this screening test is based on the fact that the larger hemoglobin molecules are precipitated by the ammonium sulfate, and myoglobin remains in the supernatant. Therefore, when myoglobin is present, the supernatant retains the red color and gives a positive reagent strip test for blood. Conversely, hemoglobin produces a red precipitate and a supernatant that tests negative for blood. Reagent strip manufacturers incorporate peroxide, and tetramethylbenzidine, into the blood testing area. Two color charts are provided that correspond to the reactions that occur with hemoglobinuria, myoglobinuria, and hematuria (RBCs). In the presence of free hemoglobin/myoglobin, uniform color ranging from a negative yellow through green to a strongly positive green-blue appears on the pad. In contrast, intact red blood cells are lysed when they come in contact with the pad, and the liberated hemoglobin produces an isolated reaction that results in a speckled pattern on the pad. Reagent strip tests can detect concentrations as low as five red blood cells per microliter; however, care must be taken when comparing these figures with the actual microscopic values, because the absorbent nature of the pad attracts some of urine. The terms trace, small, moderate, and large or trace, 1+, 2+, and 3+ are used for reporting. Reaction Interference False-positive reactions due to menstrual contamination may be seen. They also occur if strong oxidizing detergents are present in the specimen container. Vegetable peroxidase and bacterial enzymes, including an Escherichia coli peroxidase, may also cause false-positive reactions. Therefore, sediments containing bacteria should be checked closely for the presence of red blood cells. Traditionally, ascorbic acid (vitamin C) has been associated with false-negative reagent strip reactions for blood. Both Multistix and Chemstrip have modified their reagent strips to reduce this interference to very high levels (25 mg/dL) of ascorbic acid. Multistix uses a peroxide that is less subject to reduction by ascorbic acid, and Chemstrip overlays the reagent pad with an iodate-impregnated mesh that oxidizes the ascorbic acid prior to its reaching the reaction pad. False-negative reactions can result when urine with a high specific gravity Sensitivity Interference Correlations with other tests Blood Reagent Strip Multistix: Diisopropylbenzene dihydroperoxide and 3,3’,5,5’tetramethylbenzidine Chemstrip: dimethyldihydroperoxyhexane and tetramethylbenzidine Multistix: 5 to 20 RBCs/mL, 0.015 to 0.062 mg/dL hemoglobin Chemstrip: 5 RBCs/mL, hemoglobin corresponding to 10 RBCs/mL False-positive: Strong oxidizing agents Bacterial peroxidases Menstrual contamination False-negative: High specific gravity/crenated cells Formalin Captopril High concentrations of nitrite Ascorbic acid greater than 25 mg/dL Unmixed specimens Protein Microscopic contains crenated red blood cells that do not lyse when they come in contact with the reagent pad. Decreased reactivity may also be seen when formalin is used as a preservative or when the hypertension medication captopril or high concentrations of nitrite (greater than 10 mg/dL) are present. Red blood cells settle to the bottom of the specimen container, and failure to mix the specimen prior to testing causes a falsely decreased reading. Bilirubin The appearance of bilirubin in the urine can provide an early indication of liver disease. It is often detected long before the patient exhibits jaundice. Bilirubin Production Bilirubin, a highly pigmented yellow compound, is a degradation product of hemoglobin. Under normal conditions, the life span of red blood cells is approximately 120 days, at which time they are destroyed in the spleen and liver by the phagocytic cells of the reticuloendothelial system. The liberated hemoglobin is broken down into its component parts: iron, protein, and protoporphyrin. The body reuses the iron and protein, and the cells of the reticuloendothelial system convert the remaining protoporphyrin to bilirubin. The bilirubin is 3920_Ch05_071-098 23/01/14 10:15 AM Page 86 86 Part Two | Urinalysis then released into the circulation, where it binds with albumin and is transported to the liver. At this point, the kidneys cannot excrete the circulating bilirubin because not only is it bound to albumin, but it is also water insoluble (unconjugated bilirubin). In the liver, bilirubin is conjugated with glucuronic acid by the action of glucuronyl transferase to form water-soluble bilirubin diglucuronide (conjugated bilirubin). Usually, this conjugated bilirubin does not appear in the urine because it is passed directly from the liver into the bile duct and on to the intestine. In the intestine, intestinal bacteria reduce bilirubin to urobilinogen, which is then oxidized and excreted in the feces in the form of stercobilinogen and urobilin. Figure 5–3 illustrates bilirubin metabolism for reference with this section and the subsequent discussion of urobilinogen. Clinical Significance Only conjugated bilirubin can appear in the urine when the normal degradation cycle is disrupted by bile duct obstruction (post-hepatic jaundice) (e.g., gallstones or cancer) or when the integrity of the liver is damaged (hepatic jaundice), allowing leakage of conjugated bilirubin into the circulation. Hepatitis and cirrhosis are common examples of conditions that produce liver damage, resulting in bilirubinuria. Not only does the detection of urinary bilirubin provide an early indication of liver disease, but also its presence or absence can be used in determining the cause of clinical jaundice. As shown in Table 5–2, Table 5–2 Urine Bilirubin and Urobilinogen in Jaundice Bile duct obstruction Liver damage Hemolytic disease Urine Bilirubin Urine Urobilinogen +++ + or – Negative Normal ++ +++ this determination can be even more significant when bilirubin results are combined with urinary urobilinogen. Jaundice due to increased destruction of red blood cells does not produce bilirubinuria. This is because the serum bilirubin is present in the unconjugated form and the kidneys cannot excrete it. SUMMARY 5-13 Hepatitis Cirrhosis Clinical Significance of Urine Bilirubin Other liver disorders Biliary obstruction (gallstones, carcinoma) RBC Hemoglobin Ferritin Heme Globin Iron Heme oxygenase Developing erythroblasts marrow Amino acid pool Biliverdin Bilirubin (unconjugated) Plasma Unconjugated bilirubin albumin CO Bilirubin Glucuronyl transferase + Liver Glucuronic acid Lungs Kidney Bilirubin diglucuronide (conjugated) Bile ducts Urobilinogen Enterohepatic circulation Urine Urobilinogen Bacterial enzymes Stercobilinogen Fecal urobilin Figure 5–3 Hemoglobin degradation and production of bilirubin and urobilinogen. 3920_Ch05_071-098 23/01/14 10:15 AM Page 87 Chapter 5 | Chemical Examination of Urine 87 Reagent Strip (Diazo) Reactions SUMMARY 5-14 Routine testing for urinary bilirubin by reagent strip uses the diazo reaction. Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to produce an azodye, with colors ranging from increasing degrees of tan or pink to violet, respectively. Qualitative results are reported as negative, small, moderate, or large, or as negative, 1+, 2+, or 3+. Reagent strip color reactions for bilirubin are more difficult to interpret than other reagent strip reactions and are easily influenced by other pigments present in the urine. Atypical color reactions are frequently noted on visual examination and are measured by automated readers. Further testing should be performed on any questionable results. Bilirubin glucuronide + diazonium salt Acid Reagents Sensitivity Interference azodye Reaction Interference As discussed previously, false-positive reactions are primarily due to urine pigments. Of particular concern are the yelloworange urines from persons taking phenazopyridine compounds, because the thick pigment produced may be mistaken for bilirubin on initial examination. The presence of indican and metabolites of the medication Lodine may cause falsepositive readings. The false-negative results caused by the testing of specimens that are not fresh are the most frequent errors associated with bilirubin testing. Bilirubin is an unstable compound that is rapidly photo-oxidized to biliverdin when exposed to light. Biliverdin does not react with diazo tests. False-negative results also occur when hydrolysis of bilirubin diglucuronide produces free bilirubin, because this is less reactive in the reagent strip tests. High concentrations of ascorbic acid (greater than 25 mg/dL) and nitrite may lower the sensitivity of the test, because they combine with the diazonium salt and prevent its reaction with bilirubin. Ictotest Tablets A confirmatory test for bilirubin is the Ictotest (Siemens Healthcare Diagnostics Inc., Deerfield, IL). Ictotest kits consist of testing mats and tablets containing p-nitrobenzenediazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid. Ten drops of urine are added to the mat, which has special properties that cause bilirubin to remain on the surface as the urine is absorbed. Following the chemical reaction, a blue-to-purple color appears on the mat when bilirubin is present. Colors other than blue or purple appearing on the mat are considered to be a negative result. If interference in the Ictotest is suspected, it can usually be removed by adding water directly to the mat after the urine has been added. Interfering substances are washed into the mat, and only bilirubin remains on the surface. Correlations with other tests Bilirubin Reagent Strip Multistix: 2,4-dichloroaniline diazonium salt Chemstrip: 2,6-dichlorobenzenediazonium salt Multistix: 0.4 to 0.8 mg/dL bilirubin Chemstrip: 0.5 mg/dL bilirubin False-positive: Highly pigmented urines, phenazopyridine Indican (intestinal disorders) Metabolites of Lodine False-negative: Specimen exposure to light Ascorbic acid greater than 25 mg/dL High concentrations of nitrite Urobilinogen PROCEDURE 5-5 Ictotest Procedure 1. Place 10 drops of urine onto one square of the absorbent test mat. 2. Using forceps, remove one Ictotest reagent tablet, recap the bottle promptly, and place the tablet in the center of the moistened area. 3. Place 1 drop of water onto the tablet and wait 5 seconds. 4. Place a second drop of water onto the tablet so that the water runs off the tablet onto the mat. 5. Observe the color of the mat around the tablet at the end of 60 seconds. The presence of a blue-to-purple color on the mat indicates that bilirubin is present. A slight pink or red color should be ignored. Report as positive or negative. Urobilinogen As shown in Figure 5–2, when conjugated bilirubin is excreted through the bile duct into the intestine, the intestinal bacteria convert the bilirubin to a combination of urobilinogen and stercobilinogen. Some of the urobilinogen is reabsorbed from the intestine into the blood, recirculates to the liver, and is excreted back into the intestine through the bile duct. The stercobilinogen cannot be reabsorbed and remains in the intestine where it is oxidized to stercobilin. The recirculated urobilinogen that reaches the intestine is also oxidized to urobilin. Both stercobilin and 3920_Ch05_071-098 23/01/14 10:15 AM Page 88 88 Part Two | Urinalysis urobilin are excreted in the feces and are the pigments responsible for the characteristic brown color of feces. Urobilinogen appears in the urine because, as it circulates in the blood back to the liver, it passes through the kidney and is filtered by the glomerulus. Therefore, a small amount of urobilinogen—less than 1 mg/dL or Ehrlich unit—is normally found in the urine. Clinical Significance Increased urine urobilinogen (greater than 1 mg/dL) is seen in liver disease and hemolytic disorders. Measurement of urine urobilinogen can be valuable in the detection of early liver disease; however, studies have shown that when urobilinogen tests are routinely performed, 1% of the nonhospitalized population and 9% of a hospitalized population exhibit elevated results.13 This is frequently caused by constipation. Impairment of liver function decreases the ability of the liver to process the urobilinogen recirculated from the intestine. The excess urobilinogen remaining in the blood is filtered by the kidneys and appears in the urine. The clinical jaundice associated with hemolytic disorders results from the increased amount of circulating unconjugated bilirubin. This unconjugated bilirubin is presented to the liver for conjugation, resulting in a markedly increased amount of conjugated bilirubin entering the intestines. As a result, increased urobilinogen is produced, and increased amounts of urobilinogen are reabsorbed into the blood and circulated through the kidneys where filtration takes place. In addition, the overworked liver does not process the reabsorbed urobilinogen as efficiently, and additional urobilinogen is presented for urinary excretion. Although it cannot be determined by reagent strip, the absence of urobilinogen in the urine and feces is also diagnostically significant and represents an obstruction of the bile duct that prevents the normal passage of bilirubin into the intestine. An additional observation is the production of pale stools as the result of the lack of urobilin. See Table 5–2 for an outline of the relationship of urine bilirubin and urine urobilinogen to the pathologic conditions associated with them. Reagent Strip Reactions and Interference The reagent strip reactions for urobilinogen differ between Multistix and Chemstrip much more significantly than do other reagent strip parameters. Multistix uses Ehrlich’s aldehyde reaction, in which urobilinogen reacts with p-dimethylaminobenzaldehyde (Ehrlich reagent) to produce colors ranging from light to dark pink. Results are reported as Ehrlich units (EU), which are equal to mg/dL, ranging from normal readings of 0.2 and 1 through abnormal readings of 2, 4, and 8. Chemstrip incorporates SUMMARY 5-15 Clinical Significance of Urine Urobilinogen Early detection of liver disease Liver disorders, hepatitis, cirrhosis, carcinoma Hemolytic disorders an azo-coupling (diazo) reaction using 4-methoxybenzenediazonium-tetrafluoroborate to react with urobilinogen, producing colors ranging from white to pink. This reaction is more specific for urobilinogen than the Ehrlich reaction. Results are reported in mg/dL. Both tests detect urobilinogen that is present in normal quantities, and color comparisons are provided for the upper limits of normal as well as abnormal concentrations. Reagent strip tests cannot determine the absence of urobilinogen, which is significant in biliary obstruction. MULTISTIX: Urobilinogen + p-dimethylaminobenzaldehyde (Ehrlich’s (Ehrlich’s reagent) reactive substances) Acid red color CHEMSTRIP: Acid Urobilinogen + diazonium salt red azodye (4-methyloxybenzene-diazonium-tetrafluoroborate) Reaction Interference The Ehrlich reaction on Multistix is subject to a variety of interferences, referred to as Ehrlich-reactive compounds that produce false-positive reactions. These include porphobilinogen, indican, p-aminosalicylic acid, sulfonamides, methyldopa, procaine, and chlorpromazine compounds. The presence of porphobilinogen is clinically significant; however, the reagent strip test is not considered a reliable method to screen for its presence. Porphobilinogen will be discussed in Chapter 8. The sensitivity of the Ehrlich reaction increases with temperature, and testing should be performed at room temperature. Highly pigmented urines cause atypical readings with both brands of reagent strips. As a result of increased excretion of bile salts, urobilinogen results are normally highest following a meal. False-negative results occur most frequently when specimens are improperly preserved, allowing urobilinogen to be photo-oxidized to urobilin. High concentrations of nitrite interfere with the azo-coupling reaction on Chemstrip. Falsenegative readings also are obtained with both strips when formalin is used as a preservative. Nitrite Clinical Significance The reagent strip test for nitrite provides a rapid screening test for the presence of urinary tract infection (UTI). The test is designed to detect cases in which the need for a culture may not TECHNICAL TIP The urobilinogen test pad on the Multistix Pro11 and Clinitek Microalbumin strips has been replaced by the protein-low test pad. 3920_Ch05_071-098 23/01/14 10:15 AM Page 89 Chapter 5 | Chemical Examination of Urine 89 SUMMARY 5-16 Reagents Sensitivity Interference Correlations with other tests Urobilinogen Reagent Strip Multistix: p-dimethylaminobenzaldehyde Chemstrip: 4-methoxybenzenediazonium-tetrafluoroborate Multistix: 0.2 mg/dL urobilinogen Chemstrip: 0.4 mg/dL urobilinogen Multistix: False-positive: Porphobilinogen Indican p-aminosalicylic acid Sulfonamides Methyldopa Procaine Chlorpromazine Highly pigmented urine False-negative: Old specimens Preservation in formalin Chemstrip: False-positive: Highly pigmented urine False-negative: Old specimens Preservation in formalin High concentrations of nitrite Bilirubin be apparent; it is not intended to replace the urine culture as the primary test for diagnosing and monitoring bacterial infection. Many UTIs are believed to start in the bladder as a result of external contamination and, if untreated, progress upward through the ureters to the tubules, renal pelvis, and kidney. The nitrite test is valuable for detecting initial bladder infection (cystitis), because patients are often asymptomatic or have vague symptoms that would not lead the physician to order a urine culture. Pyelonephritis, an inflammatory process of the kidney and adjacent renal pelvis, is a frequent complication of untreated cystitis and can lead to renal tissue damage, impairment of renal function, hypertension, and even septicemia. Therefore, detection of bacteriuria through the use of the nitrite screening test and subsequent antibiotic therapy can prevent these serious complications. The nitrite test also can be used to evaluate the success of antibiotic therapy and to periodically screen persons with recurrent infections, patients with diabetes, and pregnant women, all of whom are considered to be at high risk for UTI. As discussed in the following section, many laboratories use the nitrite test in combination with the leukocyte esterase test to determine the necessity of performing urine cultures. Reagent Strip Reactions The chemical basis of the nitrite test is the ability of certain bacteria to reduce nitrate, a normal constituent of urine, to nitrite, which does not normally appear in the urine. Nitrite is detected by the Greiss reaction, in which nitrite at an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a diazonium compound that then reacts with tetrahydrobenzoquinolin compounds to produce a pink-colored azodye. To prevent false-positive reactions in externally contaminated specimens, the sensitivity of the test is standardized to correspond with a quantitative bacterial culture criterion of 100,000 organisms per milliliter. Although different shades of pink may be produced, the test does not measure the degree of bacteriuria, and any shade of pink is considered to represent a clinically significant amount of bacteria. Results are reported only as negative or positive. Acid Para-arsanilic acid or sulfanilamide + NO2 diazonium salt (nitrite) Acid Diazonium salt + tetrahydrobenzoquinolin pink azodye Reaction Interference Several major factors can influence the reliability of the nitrite test, and tests with negative results in the presence of even vaguely suspicious clinical symptoms should always be repeated or followed by a urine culture. 1. Bacteria that lack the enzyme reductase do not possess the ability to reduce nitrate to nitrite. Reductase is found in the gram-negative bacteria (Enterobacteriaceae) that most frequently cause UTIs. Non–nitrate-reducing grampositive bacteria and yeasts, however, cause a significant number of infections, and the nitrite test does not detect the presence of these organisms. SUMMARY 5-17 Clinical Significance of Urine Nitrite Cystitis Pyelonephritis Evaluation of antibiotic therapy Monitoring of patients at high risk for urinary tract infection Screening of urine culture specimens 3920_Ch05_071-098 23/01/14 10:15 AM Page 90 90 Part Two | Urinalysis 2. Bacteria capable of reducing nitrate must remain in contact with the urinary nitrate long enough to produce nitrite. Therefore, nitrite tests should be performed on first morning specimens or specimens collected after urine has remained in the bladder for at least 4 hours. The correlation between positive cultures and positive nitrite test results is significantly lower when testing is performed on random samples.2 3. The reliability of the test depends on the presence of adequate amounts of nitrate in the urine. This is seldom a problem in patients on a normal diet that contains green vegetables; however, because diet usually is not controlled prior to testing, the possibility of a false-negative result owing to lack of dietary nitrate does exist. 4. Further reduction of nitrite to nitrogen may occur when large numbers of bacteria are present, and this causes a false-negative reaction. 5. Other causes of false-negative results include inhibition of bacterial metabolism by the presence of antibiotics, large quantities of ascorbic acid interfering with the diazo reaction, and decreased sensitivity in specimens with a high specific gravity. Large amounts of ascorbic acid compete with nitrite to combine with the diazonium salt, therefore preventing a true nitrite measurement. SUMMARY 5-18 Reagents Sensitivity Interference Correlations with other tests Nitrite Reagent Strip Multistix: p-arsanilic acid Tetrahydrobenzo(h)-quinolin-3-ol Chemstrip: Sulfanilamide, hydroxytetrahydro benzoquinoline Multistix: 0.06 to 0.1 mg/dL nitrite ion Chemstrip: 0.05 mg/dL nitrite ion False-negative: Nonreductase-containing bacteria Insufficient contact time between bacteria and urinary nitrate Lack of urinary nitrate Large quantities of bacteria converting nitrite to nitrogen Presence of antibiotics High concentrations of ascorbic acid High specific gravity False-positive: Improperly preserved specimens Highly pigmented urine Protein Leukocytes Microscopic Leukocyte Esterase Prior to the development of the reagent strip leukocyte esterase (LE) test, detection of increased urinary leukocytes required microscopic examination of the urine sediment. This can be subject to variation depending on the method used to prepare the sediment and the technical personnel examining the sediment. Therefore, the chemical test for leukocytes offers a more standardized means for the detection of leukocytes. The test is not designed to measure the concentration of leukocytes, and the manufacturers recommend that quantitation be done by microscopic examination. An additional advantage to the chemical LE test is that it detects the presence of leukocytes that have been lysed, particularly in dilute alkaline urine, and would not appear in the microscopic examination. Clinical Significance Normal values for leukocytes are based on the microscopic sediment examination and vary from 0 to 2 to 0 to 5 per highpower field. Women tend to have higher numbers than men as a result of vaginal contamination. Increased urinary leukocytes are indicators of UTI. The LE test detects the presence of esterase in the granulocytic white blood cells (neutrophils, eosinophils, and basophils) and monocytes, but not lymphocytes. Neutrophils are the leukocytes most frequently associated with bacterial infections. Esterases also are present in Trichomonas and histiocytes. Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain esterases. A positive LE test result is most frequently accompanied by the presence of bacteria, which, as discussed previously, may or may not produce a positive nitrite reaction. Infections caused by Trichomonas, Chlamydia, yeast, and inflammation of renal tissues (i.e., interstitial nephritis) produce leukocyturia without bacteriuria. Screening urine specimens using the LE and nitrite chemical reactions to determine the necessity of performing urine cultures can be a cost-effective measure.14 The LE test contributes significantly more to the reliability of this practice than does the nitrite test. Reagent Strip Reaction The reagent strip reaction uses the action of LE to catalyze the hydrolysis of an acid ester embedded on the reagent pad to produce an aromatic compound and acid. The aromatic SUMMARY 5-19 Clinical Significance of Urine Leukocytes Bacterial and nonbacterial urinary tract infection Inflammation of the urinary tract Screening of urine culture specimens 3920_Ch05_071-098 23/01/14 10:15 AM Page 91 Chapter 5 | Chemical Examination of Urine 91 compound then combines with a diazonium salt present on the pad to produce a purple azodye. Indoxylcarbonic acid ester + diazonium salt Leukocyte Esterase Acid indoxyl + acid indoxyl purple azodye The LE reaction requires the longest time of all the reagent strip reactions (2 minutes). Reactions are reported as trace, small, moderate, and large or trace, 1+, 2+, and 3+. Trace readings may not be significant and should be repeated on a fresh specimen. Reaction Interference The presence of strong oxidizing agents or formalin in the collection container causes false-positive reactions. Highly pigmented urines and the presence of the antibiotic nitrofurantoin may obscure the color reaction. False-negative results may occur in the presence of high concentrations of protein (greater than 500 mg/dL), glucose (greater than 3 g/dL), oxalic acid, and ascorbic acid. In this reaction, ascorbic acid also combines with the diazonium salt. Crenation of leukocytes preventing release of esterases may occur in urines with a high specific gravity. The presence of the antibiotics gentamicin, cephalexin, cephalothin, and tetracycline decreases the sensitivity of the reaction. SUMMARY 5-20 Reagents Sensitivity Interference Correlations with other tests Leukocyte Esterase Reagent Strip Multistix: Derivatized pyrrole amino acid ester Diazonium salt Chemstrip: Indoxylcarbonic acid ester Diazonium salt Multistix: 5 to 15 WBC/hpf Chemstrip: 10 to 25 WBC/hpf False-positive: Strong oxidizing agents Formalin Highly pigmented urine, nitrofurantoin False-negative: High concentrations of protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines; inaccurate timing Protein Nitrite Microscopic Specific Gravity The reagent strip test for specific gravity is included as part of the physical examination of urine in Chapter 4 and is reviewed here as part of the chemical examination. A summary of the clinical significance is included in this chapter. Reagent Strip Reaction The reagent strip reaction is based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium. The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number of ions in the solution. The higher the concentration of urine, the more hydrogen ions are released, thereby lowering the pH. Incorporation of the indicator bromthymol blue on the reagent pad measures the change in pH. As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow 1.030 [acid]). Readings can be made in 0.005 intervals by careful comparison with the color chart. The specific gravity reaction is diagrammed in Figure 5–4. SUMMARY 5-21 Clinical Significance of Urine Specific Gravity Monitoring patient hydration and dehydration Loss of renal tubular concentrating ability Diabetes insipidus Determination of unsatisfactory specimens due to low concentration COOH COOH COOH COOH COOH COOH POLYELECTROLYTE ON REAGENT STRIP + + – – + – + – – + + – – + – + Ions in urine with low specific gravity Ions in urine with high specific gravity COOH COO–H+ COOH COOH COO–H+ COOH COO–H+ COO–H+ COO–H+ COO–H+ COO–H+ COO–H+ INITIAL REACTION ON REAGENT STRIP 2H+ - Bromothymol blue 6H+ - Bromothymol blue Blue-green alkaline pH Yellow-green acid pH SECONDARY REACTION ON REAGENT STRIP Figure 5–4 Diagram of reagent strip–specific gravity reaction. 3920_Ch05_071-098 23/01/14 10:16 AM Page 92 92 Part Two | Urinalysis Reaction Interference The reagent strip specific gravity measures only ionic solutes, thereby eliminating the interference by the large organic molecules, such as urea and glucose, and by radiographic contrast media and plasma expanders that are included in physical measurements of specific gravity. This difference must be considered when comparing specific gravity results obtained by a different method. Elevated concentrations of protein slightly increase the readings as a result of protein anions. Specimens with a pH of 6.5 or higher have decreased readings caused by interference with the bromthymol blue indicator (the blue-green readings associated with an alkaline pH correspond to a low specific gravity reading). Therefore, manufacturers recommend adding 0.005 to specific gravity readings when the pH is 6.5 or higher. The correction is performed by automated strip readers. SUMMARY 5-22 Reagents Sensitivity Interference Urine Specific Gravity Reagent Strip Multistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue Chemstrip: Ethylene glycol diaminoethyl ether tetraacetic acid, bromthymol blue 1.000 to 1.030 False-positive: High concentrations of protein False-negative: Highly alkaline urines (greater than 6.5) Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Chemstrip 10UA product Insert, Roche Diagnostics, Indianapolis, IN, 2004. 2. Multistix Pro Reagent Strips Product Insert. Siemens Diagnostics, Tarrytown, NY 2005. 3. TechniTips, Miles Diagnostics, Elkhart, IN, October, 1992. 4. Clinical and Laboratory Standards Institute Approved Guideline GP16-A3: Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline, ed 3, CLSI, Wayne, PA, 2009. 5. College of American Pathologists, CAP Today, Confirmatory testing. Chicago, IL. December 2011. 6. Tips from the Clinical Experts, Medical Laboratory Observer. Web site: http://www.mlo-online.com/articles/mlo0802tips.htm 7. Pugia, MJ, and Lott, JA: New developments in urinalysis strip tests for proteins. In Bayer Encyclopedia of Urinalysis. Bayer Diagnostics, Elkhart, IN, 2002. 8. Bhuwnesh, A, et al: Microalbumin screening by reagent strip predicts cardiovascular risk in hypertension. J Hypertens 14: 223–228, 1992. 9. Bianchi, S, et al: Microalbuminurea in essential hypertension. J Nephrol 10(4):216–219, 1997. 10. Clinitek Microalbumin Reagent Strip Product Insert. Bayer Diagnostics, Elkhart, IN, 2006. 11. Benedict, SR: A reagent for the detection of reducing sugars. J Biol Chem 5:485–487, 1909. 12. Lane R, and Phillips, M: Rhabdomyolysis has many causes including statins and may be fatal. Brit J Med 327:115–116, 2003. 13. Hager, CB, and Free, AH: Urine urobilinogen as a component of routine urinalysis. Am J Med Technol 36(5):227–233, 1970. 14. Wise, KA, Sagert, LA, and Grammens, GL: Urine leukocyte esterase and nitrite tests as an aid to predict urine culture results. Lab Med 15(3):186–187, 1984. Study Questions 1. Leaving excess urine on the reagent strip after removing it from the specimen will: A. Cause run-over between reagent pads B. Alter the color of the specimen C. Cause reagents to leach from the pads D. Not affect the chemical reactions 2. Failure to mix a specimen before inserting the reagent strip will primarily affect the: A. Glucose reading B. Blood reading C. Leukocyte reading D. Both B and C 3. Testing a refrigerated specimen that has not warmed to room temperature will adversely affect: A. Enzymatic reactions B. Dye-binding reactions C. The sodium nitroprusside reaction D. Diazo reactions 4. The reagent strip reaction that requires the longest reaction time is the: A. Bilirubin B. pH C. Leukocyte esterase D. Glucose 3920_Ch05_071-098 23/01/14 10:16 AM Page 93 Chapter 5 | Chemical Examination of Urine 93 5. Quality control of reagent strips is performed: A. Using positive and negative controls 12. All of the following will cause false-positive protein reagent strip values except: B. When results are questionable A. Microalbuminuria C. At least once every 24 hours B. Highly buffered alkaline urines D. All of the above 6. All of the following are important to protect the integrity of reagent strips except: A. Removing the desiccant from the bottle B. Storing in an opaque bottle C. Storing at room temperature D. Resealing the bottle after removing a strip 7. The principle of the reagent strip test for pH is the: A. Protein error of indicators B. Greiss reaction C. Delay in removing the reagent strip from the specimen D. Contamination by quaternary ammonium compounds 13. A patient with a 2+ protein reading in the afternoon is asked to submit a first morning specimen. The second specimen has a negative protein reading. This patient is: A. Positive for orthostatic proteinuria B. Negative for orthostatic proteinuria C. Positive for Bence Jones protein D. Negative for clinical proteinuria 14. Testing for microalbuminuria is valuable for early detection of kidney disease and monitoring patients with: C. Dissociation of a polyelectrolyte A. Hypertension D. Double indicator reaction B. Diabetes mellitus 8. A urine specimen with a pH of 9.0: A. Indicates metabolic acidosis B. Should be recollected C. May contain calcium oxalate crystals D. Is seen after drinking cranberry juice 9. In the laboratory, a primary consideration associated with pH is: A. Identifying urinary crystals B. Monitoring vegetarian diets C. Determining specimen acceptability D. Both A and C 10. Indicate the source of the following proteinurias by placing a 1 for prerenal, 2 for renal, or 3 for postrenal in front of the condition. C. Cardiovascular disease risk D. All of the above 15. The primary chemical on the reagent strip in the MicralTest for microalbumin binds to: A. Protein B. Antihuman albumin antibody C. Conjugated enzyme D. Galactoside 16. All of the following are true for the ImmunoDip test for microalbumin except: A. Unbound antibody migrates farther than bound antibody B. Blue latex particles are coated with antihuman albumin antibody A. ____Microalbuminuria C. Bound antibody migrates further than unbound antibody B. ____Acute phase reactants D. It utilizes an immunochromographic principle C. ____Pre-eclampsia D. ____Vaginal inflammation 17. The principle of the protein-high pad on the Multistix Pro reagent strip is the: E. ____Multiple myeloma A. Diazo reaction F. ____Orthostatic proteinuria B. Enzymatic dye-binding reaction G. ____Prostatitis 11. The principle of the protein error of indicators reaction is that: A. Protein keeps the pH of the urine constant C. Protein error of indicators D. Microalbumin-Micral-Test 18. Which of the following is not tested on the Multistix Pro reagent strip? B. Albumin accepts hydrogen ions from the indicator A. Urobilinogen C. The indicator accepts hydrogen ions from albumin B. Specific gravity C. Creatinine D. Albumin changes the pH of the urine D. Protein-high 3920_Ch05_071-098 23/01/14 10:16 AM Page 94 94 Part Two | Urinalysis 19. The principle of the protein-low reagent pad on the Multistix Pro is the: 27. The most significant reagent strip test that is associated with a positive ketone result is: A. Binding of albumin to sulphonphthalein dye A. Glucose B. Immunologic binding of albumin to antibody B. Protein C. Reverse protein error of indicators reaction C. pH D. Enzymatic reaction between albumin and dye D. Specific gravity 20. The principle of the creatinine reagent pad on microalbumin reagent strips is the: 28. The primary reagent in the reagent strip test for ketones is: A. Glycine A. Double indicator reaction B. Lactose B. Diazo reaction C. Sodium hydroxide C. Pseudoperoxidase reaction D. Sodium nitroprusside D. Reduction of a chromogen 21. The purpose of performing an albumin:creatinine ratio is to: A. Estimate the glomerular filtration rate B. Correct for hydration in random specimens C. Avoid interference for alkaline urines D. Correct for abnormally colored urines 22. A patient with a normal blood glucose and a positive urine glucose should be further checked for: A. Diabetes mellitus B. Renal disease C. Gestational diabetes D. Pancreatitis 23. The principle of the reagent strip tests for glucose is the: 29. Ketonuria may be caused by all of the following except: A. Bacterial infections B. Diabetic acidosis C. Starvation D. Vomiting 30. Urinalysis on a patient with severe back and abdominal pain is frequently performed to check for: A. Glucosuria B. Proteinuria C. Hematuria D. Hemoglobinuria 31. Place the appropriate number or numbers in front of each of the following statements. Use both numbers for an answer if needed. A. Peroxidase activity of glucose 1. Hemoglobinuria B. Glucose oxidase reaction 2. Myoglobinuria C. Double sequential enzyme reaction A. ____ Associated with transfusion reactions D. Dye-binding of glucose and chromogen B. ____ Clear red urine and pale yellow plasma 24. All of the following may produce false-negative glucose reactions except: A. Detergent contamination C. ____ Clear red urine and red plasma D. ____ Associated with rhabdomyolysis B. Ascorbic acid E. ____ Produces hemosiderin granules in urinary sediments C. Unpreserved specimens F. ____Associated with acute renal failure D. Low urine temperature 32. The principle of the reagent strip test for blood is based on the: 25. The primary reason for performing a Clinitest is to: A. Check for high ascorbic acid levels A. Binding of heme and a chromogenic dye B. Confirm a positive reagent strip glucose B. Peroxidase activity of heme C. Check for newborn galactosuria C. Reaction of peroxide and chromogen D. Confirm a negative glucose reading D. Diazo activity of heme 26. The three intermediate products of fat metabolism include all of the following except: 33. A speckled pattern on the blood pad of the reagent strip indicates: A. Acetoacetic acid A. Hematuria B. Ketoacetic acid B. Hemoglobinuria C. β -hydroxybutyric acid C. Myoglobinuria D. Acetone D. All of the above 3920_Ch05_071-098 23/01/14 10:16 AM Page 95 Chapter 5 | Chemical Examination of Urine 95 34. List the following products of hemoglobin degradation in the correct order by placing numbers 1 to 4 in the blank. A. ____Conjugated bilirubin 42. All of the following can cause a negative nitrite reading except: A. Gram-positive bacteria B. Gram-negative bacteria B. ____Urobilinogen and stercobilinogen C. Random urine specimens C. ____Urobilin D. Heavy bacterial infections D. ____Unconjugated bilirubin 35. The principle of the reagent strip test for bilirubin is the: 43. A positive nitrite test and a negative leukocyte esterase test is an indication of a: A. Dilute random specimen A. Diazo reaction B. Specimen with lysed leukocytes B. Ehrlich reaction C. Vaginal yeast infection C. Greiss reaction D. Specimen older than 2 hours D. Peroxidase reaction 36. An elevated urine bilirubin with a normal urobilinogen is indicative of: A. Cirrhosis of the liver 44. All of the following can be detected by the leukocyte esterase reaction except: A. Neutrophils B. Eosinophils B. Hemolytic disease C. Lymphocytes C. Hepatitis D. Basophils D. Biliary obstruction 37. The primary cause of a false-negative bilirubin reaction is: A. Highly pigmented urine B. Specimen contamination C. Specimen exposure to light D. Excess conjugated bilirubin 38. The purpose of the special mat supplied with the Ictotest tablets is that: A. Bilirubin remains on the surface of the mat. B. It contains the dye needed to produce color. C. It removes interfering substances. D. Bilirubin is absorbed into the mat. 39. The reagent in the Multistix reaction for urobilinogen is: 45. Screening tests for urinary infection combine the leukocyte esterase test with the test for: A. pH B. Nitrite C. Protein D. Blood 46. The principle of the leukocyte esterase reagent strip test uses a: A. Peroxidase reaction B. Double indicator reaction C. Diazo reaction D. Dye-binding technique 47. The principle of the reagent strip test for specific gravity uses the dissociation constant of a(n): A. A diazonium salt A. Diazonium salt B. Tetramethylbenzidine B. Indicator dye C. p-Dimethylaminobenzaldehyde C. Polyelectrolyte D. Hoesch reagent D. Enzyme substrate 40. The primary problem with urobilinogen tests using Ehrlich reagent is: 48. A specific gravity of 1.005 would produce the reagent strip color: A. Positive reactions with porphobilinogen A. Blue B. Lack of specificity B. Green C. Positive reactions with Ehrlich’s reactive substances C. Yellow D. All of the above D. Red 41. The reagent strip test for nitrite uses the: A. Greiss reaction 49. Reagent strip–specific gravity readings are affected by: A. Glucose B. Ehrlich reaction B. Radiographic dye C. Peroxidase reaction C. Alkaline urine D. Pseudoperoxidase reaction D. All of the above 3920_Ch05_071-098 23/01/14 10:16 AM Page 96 96 Part Two | Urinalysis Case Studies and Clinical Situations 1. A patient taken to the emergency department after an episode of syncope has a fasting blood glucose level of 450 mg/dL. Results of the routine urinalysis are as follows: COLOR: Yellow KETONES: 2+ CLARITY: Clear BLOOD: Negative SP. GRAVITY: 1.015 BILIRUBIN: Negative pH: 5.0 PROTEIN-LOW: 15 mg/dL PROTEIN-HIGH: 30 mg/dL NITRITE: Negative GLUCOSE: 250 mg/dL LEUKOCYTES: Negative CREATININE: 200 mg/dL a. Explain the correlation between the patient’s blood and urine glucose results. b. What is the most probable metabolic disorder associated with this patient? c. Considering the patient’s condition, what is the significance of the patient’s protein to creatinine ratio result? d. If the patient in this study had a normal blood glucose level and normal protein and creatinine results, to what would the urinary glucose be attributed? 2. Results of a urinalysis performed on a patient scheduled for gallbladder surgery are as follows: SP. GRAVITY: 1.020 BILIRUBIN: Negative pH: 6.0 UROBILINOGEN: 8 EU PROTEIN: Negative NITRITE: Negative GLUCOSE: Negative LEUKOCYTES: Negative a. Would these results be indicative of hematuria or hemoglobinuria? b. Correlate the patient’s condition with the urobilinogen result. c. Why is the urine bilirubin result negative in this jaundiced patient? d. Would this method also measure urine porphyrins? Why or why not? 4. A female patient arrives at the outpatient clinic with symptoms of lower back pain and urinary frequency with a burning sensation. She is a firm believer in the curative powers of vitamins. She has tripled her usual dosage of vitamins in an effort to alleviate her symptoms; however, the symptoms have persisted. She is given a sterile container and asked to collect a midstream clean-catch urine specimen. Results of this routine urinalysis are as follows: COLOR: Dark yellow KETONES: Negative CLARITY: Hazy BLOOD: Negative SP. GRAVITY: 1.012 BILIRUBIN: Negative COLOR: Amber KETONES: Negative pH: 7.0 UROBILINOGEN: Normal CLARITY: Hazy BLOOD: Negative PROTEIN: Trace NITRITE: Negative SP. GRAVITY: 1.022 BILIRUBIN: Moderate GLUCOSE: Negative LEUKOCYTES: 1+ pH: 6.0 UROBILINOGEN: Normal Microscopic PROTEIN: Negative NITRITE: Negative 8 TO 12 RBC/HPF GLUCOSE: Negative LEUKOCYTES: Negative 40 TO 50 WBC/HPF Moderate squamous epithelial cells a. What discrepancies between the chemical and microscopic test results are present? State and explain a possible reason for each discrepancy. b. What additional chemical tests could be affected by the patient’s vitamin dosage? Explain the principle of the interference. c. Discuss the urine color and specific gravity results with regard to correlation and give a possible cause for any discrepancy. d. State three additional reasons not previously given for a negative nitrite test in the presence of increased bacteria. a. What would be observed if this specimen were shaken? b. Explain the correlation between the patient’s scheduled surgery and the normal urobilinogen. c. If blood were drawn from this patient, how might the appearance of the serum be described? d. What special handling is needed for serum and urine specimens from this patient? 3. Results of a urinalysis on a very anemic and jaundiced patient are as follows: COLOR: Red KETONES: Negative CLARITY: Clear BLOOD: Large Heavy bacteria 3920_Ch05_071-098 23/01/14 10:16 AM Page 97 Chapter 5 | Chemical Examination of Urine 97 5. Results of a urinalysis collected following practice from a 20-year-old college athlete are as follows: SP. GRAVITY: 1.017 BILIRUBIN: Negative pH: 6.5 UROBILINOGEN: 0.4 EU COLOR: Dark yellow KETONES: Negative PROTEIN: Trace NITRITE: Negative CLARITY: Hazy BLOOD: 1+ GLUCOSE: Negative LEUKOCYTES: Negative SP. GRAVITY: 1.029 BILIRUBIN: Negative pH: 6.5 UROBILINOGEN: 1 EU a. Would hematuria be suspected in this specimen? Why or why not? PROTEIN: 2+ NITRITE: Negative b. What is the most probable cause of the positive blood reaction? GLUCOSE: Negative LEUKOCYTES: Negative c. What is the source of the substance causing the positive blood reaction and the name of the condition? The physician requests that the athlete collect another specimen in the morning prior to classes and practice. a. What is the purpose of the second sample? b. What changes would you expect in the second sample? c. Is the proteinuria present in the first sample of prerenal, renal, or postrenal origin? 6. A construction worker is pinned under collapsed scaffolding for several hours prior to being taken to the emergency room. His abdomen and upper legs are severely bruised, but no fractures are detected. A specimen for urinalysis obtained by catheterization has the following results: COLOR: Red-brown KETONES: Negative CLARITY: Clear BLOOD: 4+ d. Would this patient be monitored for changes in renal function? Why or why not? 7. Considering the correct procedures for care, technique, and quality control for reagent strips, state a possible cause for each of the following scenarios. a. The urinalysis supervisor notices that an unusually large number of reagent strips are becoming discolored before the expiration date has been reached. b. A physician’s office is consistently reporting positive nitrite test results with negative LE test results. c. A student’s results for reagent strip blood and LE are consistently lower than those of the laboratory staff. d. One morning the urinalysis laboratory was reporting results that were frequently questioned by physicians. 3920_Ch05_071-098 23/01/14 10:16 AM Page 98 3920_Ch06_099-146 23/01/14 10:18 AM Page 99 CHAPTER 6 Microscopic Examination of Urine LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 6-1 List the physical and chemical parameters included in macroscopic urine screening, and state their significance. 6-2 Discuss the advantages of commercial systems over the glass-slide method for sediment examination. 6-3 Describe the recommended methods for standardizing specimen preparation and volume; centrifugation; sediment preparation, volume, and examination; and reporting results. 6-4 State the purpose of Sternheimer-Malbin, acetic acid, toluidine blue, Sudan III, Gram, Hansel, and Prussian blue stains in the examination of urine sediment. 6-5 Identify specimens that should be referred for cytodiagnostic testing. 6-6 Describe the basic principles of bright-field, phasecontrast, polarizing, dark-field, fluorescence, and interference-contrast microscopy and their relationship to sediment examination. 6-9 Discuss the significance of white blood cells (WBCs) in urine sediment. 6-10 Name, describe, and give the origin and significance of the three types of epithelial cells found in urine sediment. 6-11 Discuss the significance of oval fat bodies. 6-12 Describe the process of cast formation. 6-13 Describe and discuss the significance of hyaline, RBC, WBC, bacterial, epithelial cell, granular, waxy, fatty, and broad casts. 6-14 List and identify the normal crystals found in acidic urine. 6-15 List and identify the normal crystals found in alkaline urine. 6-16 Describe and state the significance of cystine, cholesterol, leucine, tyrosine, bilirubin, sulfonamide, radiographic dye, and ampicillin crystals. 6-7 Differentiate between normal and abnormal sediment constituents. 6-17 Differentiate between actual sediment constituents and artifacts. 6-8 Discuss the significance of red blood cells (RBCs) in urine sediment. 6-18 Correlate physical and chemical urinalysis results with microscopic observations and recognize discrepancies. KEY TERMS Birefringent Dark-field microscopy Phase-contrast microscopy Bright-field microscopy Fluorescence microscopy Polarizing microscopy Casts Interference-contrast microscopy Resolution Cylindruria Köhler illumination Uromodulin 3920_Ch06_099-146 23/01/14 10:18 AM Page 100 100 Part Two | Urinalysis The third part of routine urinalysis, after physical and chemical examination, is the microscopic examination of the urinary sediment. Its purpose is to detect and to identify insoluble materials present in the urine. The blood, kidney, lower genitourinary tract, and external contamination all contribute formed elements to the urine. These include red blood cells (RBCs), white blood cells (WBCs), epithelial cells, casts, bacteria, yeast, parasites, mucus, spermatozoa, crystals, and artifacts. Because some of these components are of no clinical significance and others are considered normal unless they are present in increased amounts, examination of the urinary sediment must include both identification and quantitation of the elements present. Microscopic analysis is subject to several procedural variations, including the methods by which the sediment is prepared, the volume of sediment actually examined, the methods and equipment used to obtain visualization, and the manner in which the results are reported. Protocols have been developed to increase the standardization and costeffectiveness of microscopic urinalysis, and they are discussed in this chapter. Macroscopic Screening To enhance the cost-effectiveness of urinalysis, many laboratories have developed protocols whereby microscopic examination of the urine sediment is performed only on specimens meeting specified criteria. Abnormalities in the physical and chemical portions of the urinalysis play a primary role in the decision to perform a microscopic analysis, thus the use of the term “macroscopic screening.” Parameters considered significant vary among laboratories but usually include color, clarity, blood, protein, nitrite, leukocyte esterase, and possibly glucose. Laboratory-designated criteria can also be programed into automated instruments. Table 6–1 illustrates the significance of these parameters. Percentages of abnormal specimens that would go undetected using these parameters differ significantly among studies. 1,2 Table 6–1 Macroscopic Screening and Microscopic Correlations Screening Test Significance Color Clarity Blood Hematuria versus hemoglobinuria/ myoglobinuria Confirm pathologic or nonpathologic cause of turbidity RBCs, RBC casts Casts, cells Bacteria, WBCs WBCs, WBC casts, bacteria Yeast Blood Protein Nitrite Leukocyte esterase Glucose The patient population must also be considered when developing protocols for macroscopic screening. Populations that have come under consideration include pregnant women, as well as pediatric, geriatric, diabetic, immunocompromised, and renal patients. The Clinical and Laboratory Standards Institute (CLSI) recommends that microscopic examination be performed when requested by a physician, when a laboratoryspecified patient population is being tested, or when any abnormal physical or chemical result is obtained.3 Specimen Preparation Specimens should be examined while fresh or adequately preserved. Formed elements—primarily RBCs, WBCs, and hyaline casts—disintegrate rapidly, particularly in dilute alkaline urine. Refrigeration may cause precipitation of amorphous urates and phosphates and other nonpathologic crystals that can obscure other elements in the urine sediment. Warming the specimen to 37°C prior to centrifuging may dissolve some of these crystals. The midstream clean-catch specimen minimizes external contamination of the sediment. As with the physical and chemical analyses, dilute random specimens may cause false-negative readings. Care must be taken to thoroughly mix the specimen prior to decanting a portion into a centrifuge tube. Specimen Volume A standard amount of urine, usually between 10 and 15 mL, is centrifuged in a conical tube. This provides an adequate volume from which to obtain a representative sample of the elements present in the specimen. A 12-mL volume is frequently used because multiparameter reagent strips are easily immersed in this volume, and capped centrifuge tubes are often calibrated to this volume. If obtaining a 12-mL specimen is not possible, as with pediatric patients, the volume of the specimen used should be noted on the report form. This allows the physician to correct the results, if indicated. Some laboratories choose to make this correction prior to reporting. For example, if 6 mL of urine is centrifuged, the results are multiplied by 2. Centrifugation The speed of the centrifuge and the length of time the specimen is centrifuged should be consistent. Centrifugation for 5 minutes at a relative centrifugal force (RCF) of 400 produces an optimum amount of sediment with the least chance of damaging the elements. To correct for differences in the diameter of centrifuge heads, RCF rather than revolutions per minute (RPM) is used. The RPM value shown on the centrifuge tachometer can be converted to RCF using nomograms available in many laboratory manuals or by using the formula: RCF = 1.118 × 10–5 × radius in centimeters × RPM2 Centrifugation calibration should be routinely performed. Use of the braking mechanism to slow the centrifuge causes 3920_Ch06_099-146 23/01/14 10:18 AM Page 101 Chapter 6 | Microscopic Examination of Urine 101 disruption of the sediment prior to decantation and should not be used. To prevent biohazardous aerosols, all specimens must be centrifuged in capped tubes. Sediment Preparation A uniform amount of urine and sediment should remain in the tube after decantation. Volumes of 0.5 and 1.0 mL are frequently used. The volume of urine centrifuged divided by the sediment volume equals the concentration factor, which in the preceding examples are 24 and 12, respectively. The sediment concentration factor relates to the probability of detecting elements present in low quantities and is used when quantitating the number of elements present per milliliter. To maintain a uniform sediment concentration factor, urine should be aspirated off rather than poured off, unless otherwise specified by the commercial system in use. Some systems provide pipettes for this purpose. The pipettes are also used for sediment resuspension and transferring specimens to the slide. The sediment must be thoroughly resuspended by gentle agitation. This can be performed using a commercial-system pipette or by repeatedly tapping the tip of the tube with the finger. Vigorous agitation should be avoided, as it may disrupt some cellular elements. Thorough resuspension is essential to provide equal distribution of elements in the microscopic examination fields. Urisystem (ThermoFisher Scientific, Waltham, MA), Count-10 (V-Tech, Inc., Pomona, CA), Quick-Prep Urinalysis System (Globe Scientific, Paramus, NJ), CenSlide 2000 Urinalysis System (International Remote Imaging Systems, Norwood, MA), and R/S Workstations 1000, 2000, 2003 (DioSys, Waterbury, CA). The systems provide a variety of options including capped, calibrated centrifuge tubes; decanting pipettes to control sediment volume; and slides that control the amount of sediment examined, produce a consistent monolayer of sediment for examination, and provide calibrated grids for more consistent quantitation. The Cen-Slide and R/S Workstations do not require manual loading of the centrifuged specimen onto a slide and are considered closed systems that minimize exposure to the specimen. Cen-Slide provides a specially designed tube that permits direct reading of the urine sediment. The R/S Workstations consist of a glass flow cell into which urine sediment is pumped, microscopically examined, and then flushed from the system. Examining the Sediment The volume of sediment placed on the microscope slide should be consistent for each specimen. When using the conventional glass-slide method, the recommended volume is 20 µ L (0.02 mL) covered by a 22 × 22 mm glass cover slip. Allowing the specimen to flow outside of the cover slip may result in the loss of heavier elements such as casts. Commercial systems control the volume of sediment examined by providing slides with chambers capable of containing a specified volume. Care must be taken to ensure the chambers are completely filled. Product literature supplies the chamber volume, size of the viewing area, and approximate number of low-power and high-power viewing areas, based on the area of the field of view using a standard microscope. This information, together with the sediment concentration factor, is necessary to quantitate cellular elements per milliliter of urine. Microscopic examination should be performed in a consistent manner and include observation of a minimum of 10 fields under both low (10×) and high (40×) power. The slide is first examined under low power to detect casts and to ascertain the general composition of the sediment. When elements such as casts that require identification are encountered, the setting is changed to high power. If the conventional glass-slide method is being used, casts have a tendency to locate near the edges of the cover slip; therefore, low-power scanning of the cover-slip perimeter is recommended. This does not occur when using standardized commercial systems. When the sediment is examined unstained, many sediment constituents have a refractive index similar to urine. Therefore, it is essential that sediments be examined under reduced light when using bright-field microscopy. Initial focusing can be difficult with a fluid specimen, and care must be taken to ensure that the examination is being performed in the correct plane. Often an epithelial cell will be present to provide a point of reference. Focusing on artifacts should be avoided, because they are often larger than the regular sediment elements and cause the microscopist to examine objects in the wrong plane. Continuous focusing with the fine adjustment aids in obtaining a complete representation of the sediment constituents. Commercial Systems Reporting the Microscopic Examination The conventional method of placing a drop of centrifuged urine on a glass slide, adding a cover slip, and examining microscopically has been substantially improved through the use of commercial slide systems. 4 The CLSI recommends their use together with standardization of all phases of the methodology, including the conventional method, as discussed in the following sections. Systems currently available include KOVA (Hycor Biomedical, Inc., Garden Grove, CA), The terminology and methods of reporting may differ slightly among laboratories but must be consistent within a particular laboratory system. Routinely, casts are reported as the average number per low-power field (lpf) following examination of 10 fields, and RBCs and WBCs, as the average number per 10 high-power fields (hpf). Epithelial cells, crystals, and other elements are frequently reported in semiquantitative terms such as, rare, few, moderate, and many, or as 1+, 2+, 3+, and 4+, Volume of Sediment Examined 3920_Ch06_099-146 23/01/14 10:18 AM Page 102 102 Part Two | Urinalysis following laboratory format as to lpf or hpf use. Laboratories must also determine their particular reference values based on the sediment concentration factor in use. For example, Urisystem, with a concentration factor of 30, states a reference value for WBCs of zero to eight per hpf, as opposed to the conventional value of zero to five per hpf used with a concentration factor of 12. Converting the average number of elements per lpf or hpf to the number per milliliter provides standardization among the various techniques in use. Steps include the following: EXAMPLE 1. Calculating the area of an lpf or hpf for the microscope in use using the manufacturer-supplied field of view diameter and the formula πr2 = area. Diameter of hpf = 0.35 mm 3.14 × 0.1752 = 0.096 mm2 2. Calculating the maximum number of lpfs or hpfs in the viewing area. Area under a 22 mm × 22 mm cover slip = 484 mm2 484 = 5040 hpfs .096 3. Calculating the number of hpfs per milliliter of urine tested using the concentration factor and the volume of sediment examined. 5040 5040 = = 21,000 hpf/mL of urine 0.02 mL × 12 .24 4. Calculating the number of formed elements per milliliter of urine by multiplying the number of hpfs per milliliter by the average number of formed elements per field. HISTORICAL NOTE Addis Count The first procedure to standardize the quantitation of formed elements in the urine microscopic analysis was developed by Addis in 1926. The Addis count, as it is called, used a hemocytometer to count the number of RBCs, WBCs, casts, and epithelial cells present in a 12-hour specimen. Normal values have a wide range and are approximately 0 to 500,000 RBCs, 0 to 1,800,000 WBCs and epithelial cells, and 0 to 5000 hyaline casts. 5 The Addis count, which was used primarily to monitor the course of diagnosed cases of renal disease, has been replaced by various standardized commercial systems for the preparation, examination, and quantitation of formed elements in nontimed specimens. Table 6–2 Microscopic Elements RBCs WBCs Epithelial cells Casts Bacteria Routine Urinalysis Correlations Physical Chemical Exceptions Turbidity Red color Turbidity + Blood + Protein + Protein + Nitrite + LE Number Hemolysis Number Lysis Turbidity Turbidity + Protein ↑ pH Turbidity + Nitrite + Leukocytes pH Color + Bilirubin 4 WBC/hpf × 21,000 = 84,000 WBC/mL Provided the same microscope and volume of sediment examined are used, the number of lpfs and hpfs per milliliter of urine remains the same, thereby simplifying the calculation. Laboratories should evaluate the advantages and disadvantages of adding an additional calculation step to the microscopic examination. The CLSI states that all decisions with regard to reporting of the microscopic should be based on the needs of the individual laboratory. Procedures should be completely documented and followed by all personnel.3 Correlating Results Microscopic results should be correlated with the physical and chemical findings to ensure the accuracy of the report. Specimens in which the results do not correlate must be rechecked for both technical and clerical errors. Table 6–2 shows some of the more common correlations in the urinalysis; however, the amount of formed elements or chemicals must also be Crystals Number Number Number and type Number and type considered, as must the possibility of interference with chemical tests and the age of the specimen. Sediment Examination Techniques Many factors can influence the appearance of the urinary sediment, including cells and casts in various stages of development and degeneration, distortion of cells and crystals by the chemical content of the specimen, the presence of inclusions in cells and casts, and contamination by artifacts. Therefore, 3920_Ch06_099-146 23/01/14 10:18 AM Page 103 Chapter 6 | Microscopic Examination of Urine 103 identification can sometimes be difficult even for experienced laboratory personnel. Identification can be enhanced through the use of sediment stains (Table 6–3) and different types of microscopy. Sediment Stains Staining increases the overall visibility of sediment elements being examined using bright-field microscopy by changing their refractive index. As mentioned, elements such as hyaline casts have a refractive index very similar to that of urine. Staining also imparts identifying characteristics to cellular structures, such as the nuclei, cytoplasm, and inclusions. The most frequently used stain in urinalysis is the Sternheimer-Malbin stain, which consists of crystal violet and Table 6–3 safranin O.6 The stain is available commercially under a variety of names, including Sedi-Stain (Becton, Dickinson, Parsippany, NJ) and KOVA stain (Hycor Biomedical, Inc., Garden Grove, CA). Commercial brands contain stabilizing chemicals to prevent the precipitation that occurred with the original stain. The dye is absorbed well by WBCs, epithelial cells, and casts, providing clearer delineation of structure and contrasting colors of the nucleus and cytoplasm. Table 6–4 provides an example of the staining reactions as shown in the product literature. A 0.5% solution of toluidine blue, a metachromatic stain, provides enhancement of nuclear detail. It can be useful in the differentiation between WBCs and renal tubular epithelial cells and is also used in the examination of cells from other body fluids. Urine Sediment Stain Characteristics Stain Action Function Sternheimer-Malbin Identifies WBCs, epithelial cells, and casts Toluidine blue Delineates structure and contrasting colors of the nucleus and cytoplasm Enhances nuclear detail 2% acetic acid Lyses RBCs and enhances nuclei of WBCs Lipid stains: Oil Red O and Sudan III Gram stain Stain triglycerides and neutral fats orange-red Do not stain cholesterol Differentiates gram-positive and gram-negative bacteria Methylene blue and eosin Y stains eosinophilic granules Stains structures containing iron Hansel stain Prussian blue stain Table 6–4 Differentiates WBCs and renal tubular epithelial (RTE) cells Distinguishes RBCs from WBCs, yeast, oil droplets, and crystals Identify free fat droplets and lipid-containing cells and casts Identifies bacterial casts Identifies urinary eosinophils Identifies yellow-brown granules of hemosiderin in cells and casts Expected Staining Reactions of Urine Sediment Constituents Elements in Urinary Sediment Usual Distinguishing Color of Stained Elements RBCs Neutral—pink to purple Acid—pink (unstained) Alkaline—purple Nuclei Cytoplasm WBCs (dark-staining cells) Purple Purple granules Glitter cells (SternheimerMalbin positive cells) Renal tubular epithelial cells Bladder tubular epithelial cells Colorless or light blue Pale blue or gray Light shade of blue-purple Light purple Dark shade of blue-purple Blue-purple Comments Some glitter cells exhibit brownian movement Continued 3920_Ch06_099-146 23/01/14 10:18 AM Page 104 104 Part Two | Urinalysis Table 6–4 Expected Staining Reactions of Urine Sediment Constituents—cont’d Elements in Urinary Sediment Usual Distinguishing Color of Stained Elements Squamous epithelial cells Dark shade of orange-purple Comments Light purple or blue Inclusions and Matrix Hyaline casts Pale pink or pale purple Coarse granular inclusion casts Finely granular inclusion casts Dark purple granules in purple matrix Fine dark purple granules in pale pink or pale purple matrix Pale pink or pale purple Waxy casts Fat inclusion casts Fat globules unstained in a pink matrix Red cell inclusion casts Pink to orange-red Blood (hemoglobin) casts Bacteria Trichomonas vaginalis Orange-red Motile: do not stain Nonmotile: stain purple Light blue-green Mucus Background Pale pink or pale blue Pale pink or pale purple Very uniform color; slightly darker than mucous threads Darker than hyaline casts, but of a pale even color; distinct broken ends Rare; presence is confirmed if examination under polarized light indicates double refraction Intact cells can be seen in matrix No intact cells Motile organisms are not impaired Motility is unimpaired in fresh specimens when recommended volumes of stain are used; immobile organisms also identifiable From Product Profile: Sedi-Stain. Clay Adams, Division of Becton, Dickinson & Company, Parsippany, NJ, 1974, with permission. Nuclear detail is also enhanced by the addition of 2% acetic acid to the sediment. This method cannot be used for initial sediment analysis because RBCs are lysed by the acetic acid (see Fig. 6–18 later in this chapter). Lipid Stains The passage of lipids (triglycerides, neutral fats, and cholesterol) across the glomerular membrane results in the appearance of free fat droplets and lipid-containing cells and casts in the urinary sediment. The lipid stains, Oil Red O and Sudan III, and polarizing microscopy can be used to confirm the presence of these elements. Triglycerides and neutral fats stain orange-red in the presence of stain, whereas cholesterol does not stain but is capable of polarization. The three lipids are usually present concurrently in the sediment, thereby permitting use of either staining or polarization for their confirmation. Gram Stain The Gram stain is used primarily in the microbiology section for the differentiation between gram-positive (blue) and gramnegative (red) bacteria. Its role in routine urinalysis is limited to the identification of bacterial casts, which can easily be confused with granular casts. To perform Gram staining, a dried, heat-fixed preparation of the urine sediment must be used. Hansel Stain Polynuclear WBCs seen in the urinary sediment are almost always neutrophils associated with microbial infection. However, in cases of a drug-induced allergic reaction producing inflammation of the renal interstitium, eosinophils are present in the sediment. The preferred stain for urinary eosinophils is Hansel stain, consisting of methylene blue and eosin Y (Lide Labs, Inc., 3920_Ch06_099-146 23/01/14 10:18 AM Page 105 Chapter 6 | Microscopic Examination of Urine 105 Florissant, MO); however, Wright’s stain can also be used. Staining is performed on a dried smear of the centrifuged specimen or a cytocentrifuged preparation of the sediment. Table 6–5 Technique Function Prussian Blue Stain Bright-field microscopy Phase-contrast microscopy Used for routine urinalysis As discussed in Chapter 5, after episodes of hemoglobinuria, yellow-brown granules may be seen in renal tubular epithelial cells and casts or free-floating in the urine sediment. To confirm that these granules are hemosiderin, the Prussian blue stain for iron is used and stains the hemosiderin granules a blue color. Cytodiagnostic Urine Testing Although it is not a part of the routine examination of the urine sediment, the preparation of permanent slides using cytocentrifugation followed by staining with Papanicolaou stain provides an additional method for detecting and monitoring renal disease. Cytodiagnostic urine testing is frequently performed independently of routine urinalysis for detection of malignancies of the lower urinary tract. A voided first morning specimen is recommended for testing, which is performed by the cytology laboratory. Cytodiagnostic urine testing also provides more definitive information about renal tubular changes associated with transplant rejection; viral, fungal, and parasitic infections; cellular inclusions; pathologic casts; and inflammatory conditions. The urinalysis laboratory should refer specimens with unusual cellular findings to the pathologist for further examination. Microscopy Microscopic examination of urine is best performed when the laboratorian is knowledgeable about the types of microscopes available, their primary characteristics, and the proper use and maintenance of these microscopes. Bright-field microscopy is the most common type of microscopy performed in the urinalysis laboratory. Other types of microscopy that are useful for examining the urine sediment are phase contrast, polarizing, dark field, fluorescence, and interference contrast (Table 6–5). The type of microscopy used depends on the specimen type, the refractive index of the object, and the ability to image unstained living cells. All microscopes are designed to magnify small objects to such a degree that the details of their structure can be analyzed. Basically, they do this by employing a variety of lenses and light sources as described in the following section. The Microscope Essentially all types of microscopes contain a lens system, illumination system, and a body consisting of a base, body tube, and nosepiece (Fig. 6–1). Primary components of the lens system are the oculars, the objectives, and the coarse- and fineadjustment knobs. The illumination system contains the light source, condenser, and field and iris diaphragms. Objects to be examined are placed on a platform, referred to as the mechanical stage. The compound bright-field microscope is used primarily in the urinalysis laboratory and consists of a two-lens Urinalysis Microscopic Techniques Polarizing microscopy Dark-field microscopy Fluorescence microscopy Interferencecontrast Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals Aids in identification of Treponema pallidum Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye including labeled antigens and antibodies Produces a three-dimensional microscopy image and layerby-layer imaging of a specimen system combined with a light source. The first lens system is located in the objective and is adjusted to be near the specimen. The second lens system, the ocular lens, is located in the eyepiece. The path of light passes through the specimen up to the eyepiece. The oculars or eyepieces of the microscope are located at the top of the body tube. Clinical laboratory microscopes are binocular, allowing the examination to be performed using both eyes to provide more complete visualization. For optimal viewing conditions, the oculars can be adjusted horizontally to adapt to differences in interpupillary distance between operators. A diopter adjustment knob on the oculars can be rotated to compensate for variations in vision between the operators’ eyes. The oculars are designed to further magnify the object that has been enhanced by the objectives for viewing. Laboratory microscopes normally contain oculars capable of increasing the magnification 10 times (10×). The field of view is determined by the eyepiece and is the diameter of the circle of view when looking through the oculars. The field of view varies with the field number engraved on the eyepiece and the magnification of the objective. The higher the magnification, the smaller the field of view will be. In urinalysis microscopy, sediment constituents are reported as the number per microscopic field (number per hpf or lpf). Objectives are contained in the revolving nosepiece located above the mechanical stage. Objectives are adjusted to be near the specimen and perform the initial magnification of the object on the mechanical stage. The image then passes to the oculars for further resolution (ability to visualize fine details). Resolution is the ability of the lens to distinguish two 3920_Ch06_099-146 23/01/14 10:18 AM Page 106 106 Part Two | Urinalysis Interpupillary distance control Body Condenser focus knob Ocular Nosepiece Objective Course adjustment knob Fine adjustment knob Mechanical stage adjustment knobs Condenser aperture diaphragm control ring Condenser Centering screw Field diaphragm control ring Rheostat Figure 6–1 Parts of the binocular microscope. PROCEDURE 6-1 Care of the Microscope 1. Carry microscope with two hands, supporting the base with one hand. 2. Always hold the microscope in a vertical position. 3. Clean optical surfaces only with a good quality lens tissue and commercial lens cleaner. 4. Do not use the 10× and 40× objectives with oil. 5. Clean the oil immersion lens after use. 6. Always remove slides with the low-power objective raised. 7. Store the microscope with the low-power objective in position and the stage centered. small objects that are a specific distance apart. Resolving power is best when the distance between the two objects is small. It is dependent on the wavelength of light and the numerical aperture of the lens. The shorter the wavelength of light, the greater the resolving power of the microscope will be. Routinely used objectives in the clinical laboratory have magnifications of 10× (low power, dry), 40× (high power, dry), and 100× (oil immersion). The objectives used for examination of urine sediment are 10× and 40×. The final magnification of an object is the product of the objective magnification times the ocular magnification. Using a 10× ocular and a 10× objective provides a total magnification of 100× and in urinalysis is the lpf observation. The 10× ocular and the 40× objective provide a magnification of 400× for hpf observations. Objectives are inscribed with information that describes their characteristics and includes the type of objective (plan used for bright field, ph for phase contrast), magnification, numerical aperture, microscope tube length, and cover-slip thickness to be used. The numerical aperture number represents the refractive index of the material between the slide and the outer lens (air or oil) and the angle of the light passing through it. The higher the numerical aperture, the better the light-gathering capability of the lens will be, thus yielding greater resolving power. The length of the objectives attached to the nosepiece varies with magnification (length increases from 10× to 100× magnification), thereby changing the distance between the lens and the slide when they are rotated. The higher the numerical aperture, the closer the lens is to the object. Most microscopes are designed to be parfocal, indicating that they require only minimum adjustment when switching among objectives. The distance between the slide and the objective is controlled by the coarse- and fine-focusing knobs located on the body tube. Initial focusing is performed using the coarse knob that moves the mechanical stage noticeably up and down until the object comes into view. This is followed by adjustment using the fine-focusing knob to sharpen the image. When using a parfocal microscope, only the fine knob should be used for adjustment when changing magnifications. Illumination for the modern microscope is provided by a light source located in the base of the microscope. The light source is equipped with a rheostat to regulate the intensity of the light. Filters may also be placed on the light source to vary the illumination and wavelengths of the emitted light. A field diaphragm contained in the light source controls the diameter of the light beam reaching the slide and is adjusted for optimal illumination. A condenser located below the stage then focuses the light on the specimen and controls the light for uniform 3920_Ch06_099-146 23/01/14 10:18 AM Page 107 Chapter 6 | Microscopic Examination of Urine 107 illumination. The normal position of the condenser is almost completely up with the front lens of the condenser near the slide but not touching it. The condenser adjustment (focus) knob moves the condenser up and down to focus light on the object. An aperture diaphragm in the condenser controls the amount of light and the angle of light rays that pass to the specimen and lens, which affects resolution, contrast, and depth of the field of image. By adjusting the aperture diaphragm to 75% of the numerical aperture of the objective, maximum resolution is achieved. The aperture diaphragm should not be used to reduce light intensity because it decreases resolution. The microscope lamp rheostat is used for this adjustment. Köhler Illumination Two adjustments to the condenser—centering and Köhler illumination—provide optimal viewing of the illuminated field. They should be performed whenever an objective is changed. To center the condenser and obtain Köhler illumination, take the following steps: • Place a slide on the stage and focus the object using the low-power objective with the condenser raised. • Close the field diaphragm. • Lower the condenser until the edges of the field diaphragm are sharply focused. • Center the image of the field diaphragm with the condenser centering screws as shown in Figure 6–2, part A. • Open the field diaphragm until its image is at the edge of the field. • Remove an eyepiece and look down through the eyepiece tube. • Adjust the aperture diaphragm until approximately 75% of the field is visible (see Fig. 6–2, part B). • Replace the eyepiece. Additional focusing of the object should be performed using the adjustment knobs and the rheostat on the light source. Routine preventive maintenance procedures on the microscope ensure good optical performance. The microscope Field of view A Field of view Illuminated zone Field of view Illuminated zone B Figure 6–2 Centering the condenser and Köhler illumination. should always be covered when not in use to protect it from dust. If any optical surface becomes coated with dust, it should be carefully removed with a camel-hair brush. Optical surfaces should be cleaned with lens paper. Clean any contaminated lens immediately with a commercial lens cleaner. An oil immersion lens must be wiped free of oil and cleaned after each use. Fingerprints and oil smears impair the sharpness of an image. An annual professional cleaning for the microscope is recommended. Light sources are replaced as necessary. Types of Microscopy Bright-Field Microscopy Bright-field microscopy, in which objects appear dark against a light background, is most frequently used in the clinical laboratory. This technique employs the basic microscope previously described with a light source emitting light in the visible wavelength range. Use of bright-field microscopy for the examination of urine sediment can present problems when the amount of light reaching the specimen is not properly controlled. Sediment constituents with a low refractive index will be overlooked when subjected to light of high intensity. Therefore, sediments must be examined using decreased light controlled by adjusting the rheostat on the light source, not by lowering the condenser. Staining of the sediment also increases the visualization of these elements when using bright-field microscopy. Phase-Contrast Microscopy As light rays pass through an object, they are slowed in comparison to the rays passing through air (media), thereby decreasing the intensity of the light and producing contrast. This is called phase difference and is affected by the thickness of the object, refractive index, and other light-absorbance properties. The best contrast is obtained when the light that does not pass through the specimen is shifted one quarter of a wavelength and compared with the phase difference of the specimen. Phase-contrast microscopy provides this contrast. Phase-contrast microscopy is accomplished by adaptation of a bright-field microscope with a phase-contrast objective lens and a matching condenser. Two phase rings that appear as “targets” are placed in the condenser and the objective. One phase ring is placed in the condenser or below it, permitting light to only pass through the central clear circular area. A second phase-shifting ring with a central circular area that retards the light by one quarter wavelength is placed in the objective. Phase rings must match, so it is important to check that the objective and condenser mode are the same. The diameter of the rings varies with the magnification. The image has the best contrast when the background is darkest. Phase-contrast rings must be adjusted to have maximum contrast. The two rings are adjusted to make them concentric. Adjustment steps are as follows:7 • Focus the microscope in bright-field with a specimen slide. • Select a low-power phase condenser ring. 3920_Ch06_099-146 23/01/14 10:18 AM Page 108 108 Part Two | Urinalysis • Select the corresponding ring objective. • Remove an ocular, insert the adjustment telescope, and look through the telescope. • Observe the dark and light rings (annuli). • With the adjusting screw on the telescope, center the light annulus (condenser) over the dark annulus (objective) (Fig. 6–3). • Replace the ocular. Light passes to the specimen through the clear circle in the phase ring in the condenser, forming a halo of light around the specimen. The diffracted light then enters the central circle of the phase-shifting ring, and all other light is moved one quarter of a wavelength out of phase. The variations of contrast in the specimen image due to the various refractive indexes in the object are observed as the light rays merge together, enhancing visualization and detail. Phase-contrast microscopy is particularly advantageous for identifying low refractive hyaline casts or mixed cellular casts and mucous threads. Polarizing Microscopy The use of polarized light aids in the identification of crystals and lipids. Both substances have the ability to rotate the path of the unidirectional polarized light beam to produce characteristic colors in crystals and Maltese cross formation in lipids. These elements seen under polarized light microscopy are birefringent, a property indicating that the element can refract light in two dimensions at 90 degrees to each other. The halogen quartz lamp in the microscope produces light rays of many different waves. Each wave has a distinct direction and a vibration perpendicular to its direction. Normal or unpolarized light vibrates in equal intensity in all directions. Polarized light vibrates in the same plane or direction. As the light passes through a birefringent substance, it splits into two beams, one beam rotated 90 degrees to the other. Isotropic substances such as blood cells do not have this refractive property, and the light passes through unchanged. A substance that rotates the plane of polarized light 90 degrees in a clockwise direction is said to have positive birefringence. In contrast, a Condenser ring substance that rotates the plane in a counterclockwise direction has negative birefringence. Polarized light is obtained by using two polarizing filters. The light emerging from one filter vibrates in one plane, and a second filter placed at a 90-degree angle blocks all incoming light, except that rotated by the birefringent substance. The filters are in opposite directions called a “crossed configuration.” Between cross-polarizing filters, birefringent crystals are visible in characteristic patterns (Fig. 6–4). Bright-field microscopes can be adapted for polarizing microscopy. Two polarizing filters must be installed in a crossed configuration. The first filter, the polarizing filter, is placed in the condenser filter holder; the second filter, the analyzer, is placed in the head between the objectives and the ocular. The polarizing filter is rotated to allow light vibrating in one direction only to reach the object. If the object does not have birefringent properties, no light will reach the analyzer filter and the object will appear black. Refracted rays from a birefringent object will reach the analyzer, causing the object to appear white or colored against the black background. An additional filter called a red compensated polarizing filter can be added to the microscope. This filter divides the light entering the microscope into slow and fast vibrations. Crystals can be more easily identified by aligning them with the slow vibration and observing the blue or yellow color they produce (see Chapter 11). Polarizing microscopy is used in urinalysis to confirm the identification of fat droplets, oval fat bodies, and fatty casts that produce a characteristic Maltese cross pattern. Birefringent uric acid crystals can be distinguished from cystine crystals, monohydrate calcium oxalate crystals from nonpolarizing RBCs, and calcium phosphate crystals differentiated from nonpolarizing bacteria by their polarizing characteristics. Interference-Contrast Microscopy Interference-contrast microscopy provides a three-dimensional image showing very fine structural detail by splitting the light ray so that the beams pass through different areas of the specimen. The light interference produced by the varied depths of the specimen is compared, and a three-dimensional image is visualized. The advantage of interference-contrast microscopy is that an object appears bright against a dark background but without the diffraction halo associated with phase-contrast microscopy. More extensive modifications to the bright-field microscope are Birefringent specimen Phase objective ring Alignment Centering phase microscope rings Figure 6–3 Phase-contrast ring adjustment. Rotated wave Light source Polarizing filter Wave separated Polarizing filter 90° by specimen 90° orientation Figure 6–4 Diagram of polarized light. 3920_Ch06_099-146 23/01/14 10:18 AM Page 109 Chapter 6 | Microscopic Examination of Urine 109 required to perform this technique. Therefore, it is not routinely used in the urinalysis laboratory. Two types of interference-contrast microscopy are available: modulation contrast (Hoffman) and differential-interference contrast (Nomarski). Bright-field microscopes can be adapted for both methods. In the modulation-contrast microscope, a split aperture is placed below the condenser, a polarizer is placed below the split aperture, and an amplitude filter is placed in back of each objective. The modulator has three zones of light transmission: a dark zone that transmits 1% of light, a gray zone that transmits 15% of light, and a clear zone that transmits 100% of light. The polarized light rays pass through a split aperture to the various areas of the specimen and to the modulator where they are converted into the variations of light intensity to produce a three-dimensional image. The differential interference-contrast microscope uses prisms. A polarizing filter to output plane-polarized light is placed between the light source and the condenser. A two-layered Nomarski-modified Wollaston prism that separates individual rays of light into ray pairs is required. The lower Wollaston prism is built into the condenser of the microscope. The upper prism is placed between the objective and the eyepiece and recombines the rays. Above the top Wollaston prism, another polarizing filter is placed that causes wave interference to occur and produce the three-dimensional image (Fig. 6–5).8 These two types of microscopy provide layer-by-layer imaging of a specimen and enhanced detail for specimens with either a low or high refractive index. black. As the light rays pass through the specimen at oblique angles, the light scatters, diffracts, or reflects off the specimen and is captured by the objective lens. The specimen appears light against the black background or dark-field (Fig. 6–6). Fluorescence Microscopy Fluorescence microscopy is a rapidly expanding technique used in the medical field today. It is used to detect bacteria and viruses within cells and tissues through a technique called immunofluorescence. Fluorescence is the property by which some atoms absorb light at a particular wavelength and subsequently emit light of a longer wavelength, termed fluorescence lifetime. The practical application in the laboratory is that it allows the visualization of naturally fluorescent substances or those that have been stained with a fluorochrome or fluorophore (fluorescent dyes) to produce an image. The specimen is illuminated with a light of a specific wavelength. Fluorescent substances absorb the energy and emit a longer wavelength of light that is visualized with the use of special filters, called the excitation filter and the emission filter. The excitation filter selects the excitation wavelength of light from a light source. The emission filter selects a specific wavelength of emitted light from the specimen to become visible. The filters are chosen to match the excitation and emission wavelengths of the fluorophore used to label the specimen. A dichroic mirror reflects the excitation light to the specimen and transmits the emitted light to the emission filter, which is collected with the objective Dark-Field Microscopy Ocular lens Dark-field microscopy is a technique used in the clinical laboratory to enhance visualization of specimens that cannot be seen easily viewed with a bright-field microscope. It is often used for unstained specimens, and, in particular, to identify the spirochete Treponema pallidum. A bright-field microscope is easily adapted for dark-field microscopy by replacing the condenser with a dark-field condenser that contains an opaque disk. The disk blocks the light from directly entering the objective, and the field of view is Analyzer Recombined rays Objective lens Three-dimensional image Upper Wollaston prism Specimen Back focal plane of objective lens Objective Specimen Condenser lens Condenser lens Split rays Condenser focal plane Dark-field stop Lower Wollaston prism Polarizer Figure 6–5 Differential interference-contrast (Nomarski) microscopy. Light rays Figure 6–6 Dark-field microscopy. 3920_Ch06_099-146 23/01/14 10:18 AM Page 110 110 Part Two | Urinalysis and imaged by the detector (Fig. 6–7). The fluorescent substance can be observed in the fluorescent microscope as a bright object against a dark background with high contrast when ultraviolet light source is used. Powerful light sources are required and are usually either mercury or xenon arc lamps.9 Urine Sediment Constituents The normal urine sediment may contain a variety of formed elements. Even the appearance of small numbers of the usually pathologically significant RBCs, WBCs, and casts can be normal. Likewise, many routine urine specimens contain nothing more than a rare epithelial cell or mucous strand. Students often have difficulty adjusting to this, because in the classroom setting, urine sediments containing abnormalities and multiple elements are usually stressed. They must learn to trust their observations after looking at the recommended number of fields. Cellular elements are also easily distorted by the widely varying concentrations, pH, and presence of metabolites in urine, making identification more difficult. Actual normal numerical values are not clearly defined. As discussed previously, urine sediment preparation methods determine the actual concentration of the sediment and, therefore, the number of elements that may be present in a microscopic field. Commonly listed values include zero to two or three RBCs per hpf, zero to five to eight WBCs per hpf, and zero to two hyaline casts per lpf. Even these figures must be taken in context with other factors, such as recent stress and exercise, menstrual contamination, and the presence of other urine sediment constituents. To put this in better perspective, the urine sediment constituents are now discussed individually with reference to the accompanying figures. Red Blood Cells In the urine, RBCs appear as smooth, non-nucleated, biconcave disks measuring approximately 7 mm in diameter (Fig. 6–8). They must be identified using high-power (40×) objective (×400 magnification). RBCs are routinely reported as the average number seen in 10 hpfs. In concentrated (hypersthenuric) urine, the cells shrink due to loss of water and may appear crenated or irregularly shaped (Fig. 6–9). In dilute (hyposthenuria) urine, the cells absorb water, swell, and lyse rapidly, releasing their hemoglobin and leaving only the cell membrane. These large empty cells are called ghost cells and can be easily missed if specimens are not examined under reduced light. Of all the urine sediment elements, RBCs are the most difficult for students to recognize. The reasons for this include RBCs’ lack of characteristic structures, variations in size, and close resemblance to other urine sediment constituents. RBCs are frequently confused with yeast cells, oil droplets, and air bubbles. Yeast cells usually exhibit budding (Fig. 6–10). Oil droplets and air bubbles are highly refractile when the fine Ocular Barrier filter Objective Figure 6–8 Normal RBCs (×400). Specimen Condenser Mercury lamp Incoming light waves Collector Excitation filter Deflecting mirror Ultraviolet light Visible light Figure 6–7 Fluorescent microscopy. Figure 6–9 Microcytic and crenated RBCs (×100). 3920_Ch06_099-146 23/01/14 10:18 AM Page 111 Chapter 6 | Microscopic Examination of Urine 111 Figure 6–10 Yeast. The presence of budding forms aid in distinguishing from RBCs (×400). adjustment is focused up and down (Fig. 6–11); they may also appear in a different plane than other sediment constituents (Fig. 6–12). The rough appearance of crenated RBCs may resemble the granules seen in WBCs, but they are much smaller than WBCs. Should the identification continue to be doubtful, adding acetic acid to a portion of the sediment will lyse the Figure 6–11 KOVA-stained squamous epithelial cells and oil droplets (×400). Notice how the oil droplet (arrow) resembles an RBC. Figure 6–12 Air bubble. Notice no formed elements are in focus (×100). RBCs, leaving the yeast, oil droplets, and WBCs intact. Supravital staining may also be helpful. Studies have focused on the morphology of urinary RBCs as an aid in determining the site of renal bleeding. RBCs that vary in size, have cellular protrusions, or are fragmented are termed dysmorphic (Fig. 6–13) and have been associated primarily with glomerular bleeding. The number and appearance of the dysmorphic cells must also be considered, because abnormal urine concentration affects RBC appearance, and small numbers of dysmorphic cells are found with nonglomerular hematuria.10,11 Dysmorphic RBCs also have been demonstrated after strenuous exercise, indicating a glomerular origin of this phenomenon.12 The dysmorphic cell most closely associated with glomerular bleeding appears to be the acanthocyte with multiple protrusions, which may be difficult to observe under bright-field microscopy.13,14 Further analysis of sediments containing dysmorphic RBCs using Wright’s stained preparations shows the cells to be hypochromic and better delineates the presence of cellular blebs and protrusions. Clinical Significance The presence of RBCs in the urine is associated with damage to the glomerular membrane or vascular injury within the genitourinary tract. The number of cells present is indicative of the extent of the damage or injury. Patient histories often mention the presence of macroscopic versus microscopic hematuria. When macroscopic hematuria is present, the urine appears cloudy with a red to brown color. Microscopic analysis may be reported in terms of greater than 100 per hpf or as specified by laboratory protocol. Macroscopic hematuria is frequently associated with advanced glomerular damage but is also seen with damage to the vascular integrity of the urinary tract caused by trauma, acute infection or inflammation, and coagulation disorders. The observation of microscopic hematuria can be critical to the early diagnosis of glomerular disorders and malignancy of the urinary tract and to confirm the presence of renal calculi. The presence of not only RBCs but also hyaline, granular, and Figure 6–13 Dysmorphic RBCs (×400). Notice the smaller size and fragmentation. 3920_Ch06_099-146 23/01/14 10:18 AM Page 112 112 Part Two | Urinalysis RBC casts may be seen following strenuous exercise. These abnormalities are nonpathologic and disappear after rest.15 The possibility of menstrual contamination must also be considered in specimens from female patients. As discussed previously, the presence or absence of RBCs in the urine sediment cannot always be correlated with specimen color or a positive chemical test result for blood. The presence of hemoglobin that has been filtered by the glomerulus produces a red urine with a positive chemical test result for blood in the absence of microscopic hematuria. Likewise, a specimen appearing macroscopically normal may contain a small but pathologically significant number of RBCs when examined microscopically. White Blood Cells WBCs are larger than RBCs, measuring an average of about 12 mm in diameter (Fig. 6–14). The predominant WBC found in the urine sediment is the neutrophil. Neutrophils are much easier to identify than RBCs because they contain granules and multilobed nuclei (Fig. 6–15 A and B). However, they are still identified using high-power microscopy and are also reported as the average SUMMARY 6-1 A Microscopic RBCs Appearance: Sources of identification error: Reporting: Complete urinalysis correlations: Non-nucleated biconcave disks Crenated in hypertonic urine Ghost cells in hypotonic urine Dysmorphic with glomerular membrane damage Yeast cells Oil droplets Air bubbles Average number per 10 hpfs Color Reagent strip blood reaction B Figure 6–15 WBCs. A. One segmented and one nonsegmented WBC (×400). B. Notice the multilobed nucleoli (×400). number seen in 10 hpfs. Neutrophils lyse rapidly in dilute alkaline urine and begin to lose nuclear detail. Neutrophils exposed to hypotonic urine absorb water and swell. Brownian movement of the granules within these larger cells produces a sparkling appearance, and they are referred to as “glitter cells.” When stained with Sternheimer-Malbin stain, these large cells stain light blue as opposed to the violet color usually seen with neutrophils. Glitter cells are of no pathologic significance (Fig. 6–16). Eosinophils Figure 6–14 RBCs and one WBC (×400). Notice the larger size and granules in the WBC. The presence of urinary eosinophils is primarily associated with drug-induced interstitial nephritis. Small numbers of eosinophils may be seen with urinary tract infection (UTI) and renal transplant rejection. Evaluation of a concentrated, stained urine sediment is required for performing a urinary eosinophil test. Urine sediment may be concentrated by routine centrifugation alone or with cytocentrifugation. The preferred eosinophil stain is Hansel (Fig. 6–17); however, Wright’s stain can also be used. The percentage of eosinophils in 100 to 500 cells is determined. Eosinophils are not normally seen in the urine; therefore, the finding of more than 1% eosinophils is considered significant.16 3920_Ch06_099-146 23/01/14 10:18 AM Page 113 Chapter 6 | Microscopic Examination of Urine 113 Figure 6–18 WBCs with acetic acid nuclear enhancement. Notice the ameboid shape in some of the WBCs. Figure 6–16 Glitter cells (×400). Observe the very noticeable granules. Usually, fewer than five leukocytes per hpf are found in normal urine; however, higher numbers may be present in urine from females. Although leukocytes, like RBCs, may enter the urine through glomerular or capillary trauma, they are also capable of ameboid migration through the tissues to sites of infection or inflammation. An increase in urinary WBCs is called pyuria and indicates the presence of an infection or inflammation in the genitourinary system. Bacterial infections, including pyelonephritis, cystitis, prostatitis, and urethritis, are frequent causes of pyuria. However, pyuria is also present in nonbacterial disorders, such as glomerulonephritis, lupus erythematosus, interstitial nephritis, and tumors. Reporting the presence of bacteria in specimens containing leukocytes is important. Epithelial Cells Figure 6–17 Hansel-stained eosinophils (×400). It is not unusual to find epithelial cells in the urine, because they are derived from the linings of the genitourinary system. Unless they are present in large numbers or in abnormal forms, Mononuclear Cells Lymphocytes, monocytes, macrophages, and histiocytes may be present in small numbers and are usually not identified in the wet preparation urine microscopic analysis. Because lymphocytes are the smallest WBCs, they may resemble RBCs. They may be seen in increased numbers in the early stages of renal transplant rejection. Monocytes, macrophages, and histiocytes are large cells and may appear vacuolated or contain inclusions. Specimens containing an increased amount of mononuclear cells that cannot be identified as epithelial cells should be referred for cytodiagnostic urine testing. The primary concern in the identification of WBCs is the differentiation of mononuclear cells and disintegrating neutrophils from round renal tubular epithelial (RTE) cells. RTE cells are usually larger than WBCs with an eccentrically located nucleus. WBCs in the process of ameboid motion may be difficult to distinguish from epithelial cells because of their irregular shape. Supravital staining or the addition of acetic acid can be used to enhance nuclear detail (Fig. 6–18), if necessary. SUMMARY 6-2 Appearance: Sources of identification error: Microscopic WBCs Larger than RBCs Granulated, multilobed neutrophils Glitter cells in hypotonic urine Mononuclear cells with abundant cytoplasm Renal tubular epithelial cells Reporting: Average number per 10 hpfs Complete urinalysis correlations: Leukocyte esterase Nitrite Specific gravity pH 3920_Ch06_099-146 23/01/14 10:18 AM Page 114 114 Part Two | Urinalysis they represent normal sloughing of old cells. Three types of epithelial cells are seen in urine: squamous, transitional (urothelial), and renal tubular (Fig. 6–19). They are classified according to their site of origin within the genitourinary system. Squamous Epithelial Cells Squamous cells are the largest cells found in the urine sediment. They contain abundant, irregular cytoplasm and a prominent nucleus about the size of an RBC (Fig. 6–20 A and B). They are often the first structures observed when the urine sediment is examined under low-power magnification. Usually at least a few squamous epithelial cells are present in the urine sediment and can serve as a good reference for focusing of the microscope. After examination of the appropriate number of fields, squamous epithelial cells are commonly reported in terms of rare, few, moderate, or many. They are reported in terms of low-power or high-power magnification based on laboratory protocol. Difficulty identifying squamous cells is rare. However, they may occasionally appear folded, possibly resembling a cast, and will begin to disintegrate in urine that is not fresh. In urine sediments containing large amounts of squamous cells, clumps of cells may make it more difficult to enumerate smaller pathologic elements, such as RBCs and WBCs, and they should be carefully examined (Figs. 6–21, 6–22, and 6–23). Squamous epithelial cells originate from the linings of the vagina and female urethra and the lower portion of the male urethra. They represent normal cellular sloughing and have no pathologic significance. Increased amounts are more frequently seen in urine from female patients. Specimens collected using the midstream clean-catch technique contain less squamous cell contamination. A variation of the squamous epithelial cell is the clue cell, which does have pathologic significance. Clue cells are indicative of vaginal infection by the bacterium Gardnerella vaginalis. They appear as squamous epithelial cells covered with the Gardnerella coccobacillus. To be considered a clue cell, the bacteria should cover most of the cell surface and extend A B Figure 6–20 A. Squamous epithelial cells identifiable under low power (×100). B. KOVA-stained squamous epithelial cells (×400). Compare the size of the nucleus with the RBCs in Figure 6-8. RBC Transitional WBC Squamous RTE Figure 6–19 Sediment-containing squamous, caudate transitional, and RTE cells (×400). Figure 6–21 Phenazopyridine-stained sediment showing squamous epithelial cells and phenazopyridine crystals formed following refrigeration (×400). beyond the edges of the cell. This gives the cell a granular, irregular appearance. Routine testing for clue cells is performed by examining a vaginal wet preparation for the presence of the characteristic cells (see Chapter 15). However, small numbers of clue cells may be present in the urinary sediment. Microscopists should remain alert for their presence, as urinalysis may be the first test performed on the patient. 3920_Ch06_099-146 23/01/14 10:18 AM Page 115 Chapter 6 | Microscopic Examination of Urine 115 Figure 6–22 Clump of squamous epithelial cells (×400). Figure 6–23 Clump of squamous epithelial cells with folded forms (×400). Figure 6–24 Transitional epithelial cells. Figure 6–25 KOVA-stained spherical transitional epithelial cells (×400). Transitional Epithelial (Urothelial) Cells Transitional epithelial cells are smaller than squamous cells and appear in several forms, including spherical, polyhedral, and caudate (Figs. 6–24, 6–25, and 6–26). These differences are caused by the ability of transitional epithelial cells to absorb large amounts of water. Cells in direct contact with the urine absorb water, becoming spherical in form and much larger than the polyhedral and caudate cells. All forms have distinct, centrally located nuclei. Transitional cells are identified and enumerated using high-power magnification. Like squamous cells, they are usually reported as rare, few, moderate, or many following laboratory protocol. Spherical forms of transitional epithelial cells are sometimes difficult to distinguish from RTE cells. The presence of a centrally located rather than eccentrically placed nucleus, and supravital staining, can aid in the differentiation. Transitional epithelial cells originate from the lining of the renal pelvis, calyces, ureters, and bladder, and from the upper portion of the male urethra. They are usually present in small Figure 6–26 Caudate transitional epithelial cells (×400). numbers in normal urine, representing normal cellular sloughing. Increased numbers of transitional cells seen singly, in pairs, or in clumps (syncytia) are present following invasive urologic procedures such as catheterization and are of no clinical significance (Fig. 6–27). An increase in transitional cells exhibiting 3920_Ch06_099-146 23/01/14 10:18 AM Page 116 116 Part Two | Urinalysis Figure 6–27 Syncytia of transitional epithelial cells from catheterized specimen (×400). Figure 6–29 RTE cells. Oval distal convoluted tubule cells. Notice the eccentrically placed nuclei (×400). abnormal morphology such as vacuoles and irregular nuclei may be indicative of malignancy or viral infection. In such cases, the specimen should be referred to the pathologist. spherical and polyhedral transitional cells (Fig. 6–30). Because RTE cells are often present as a result of tissue destruction (necrosis), the nucleus is not easily visible in unstained sediment. Cells from the collecting duct that appear in groups of three or more are called renal fragments. They are frequently seen as large sheets of cells. PCT and DCT cells are not seen in large sheets of cells (Fig. 6–31). Renal Tubular Epithelial Cells RTE cells vary in size and shape depending on the area of the renal tubules from which they originate. The cells from the proximal convoluted tubule (PCT) are larger than other RTE cells. They tend to have a rectangular shape and are referred to as columnar or convoluted cells. The cytoplasm is coarsely granular, and the RTE cells often resemble casts. They should be closely examined for the presence of a nucleus, as a nucleus would not be present in a cast. Notice the nucleus and granules in Figure 6–28. This is a PCT. This is fine cell that has absorbed fat globules and could easily be mistaken for a granular or fatty cast. Cells from the distal convoluted tubule (DCT) are smaller than those from the PCT and are round or oval. They can be mistaken for WBCs and spherical transitional epithelial cells. Observation of the eccentrically placed round nucleus aids in differentiating them from spherical transitional cells (Fig. 6–29). Collecting duct RTE cells are cuboidal and are never round. Along with the eccentrically placed nucleus, the presence of at least one straight edge differentiates them from Figure 6–30 RTE cells, cuboidal from the collecting duct (×400). N Figure 6–28 RTE cell. Columnar proximal convoluted tubule cell with granules and attached fat globules (×400). N, nucleus. Figure 6–31 Fragment of RTE cells from the collecting duct under phase microscopy (×400). 3920_Ch06_099-146 23/01/14 10:18 AM Page 117 Chapter 6 | Microscopic Examination of Urine 117 RTE cells must be identified and enumerated using high-power magnification. Depending on laboratory protocol, they may be reported as rare, few, moderate, or many, or as the actual number per high-power field. Classification of RTE cells as to site of origin is not considered a part of the routine sediment analysis and often requires special staining techniques. The presence of more than two RTE cells per high-power field indicates tubular injury, and such specimens should be referred for cytologic urine testing.17 Clinical Significance RTE cells are the most clinically significant of the epithelial cells. The presence of increased amounts is indicative of necrosis of the renal tubules, with the possibility of affecting overall renal function. Conditions producing tubular necrosis include exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin toxicity, viral infections (hepatitis B), pyelonephritis, allergic reactions, malignant infiltrations, salicylate poisoning, and acute allogenic transplant rejection. RTE cells may also be seen as secondary effects of glomerular disorders. Renal fragments are an indication of severe tubular injury with basement membrane disruption. Single cuboidal cells are particularly noticeable in cases of salicylate poisoning. Because one of the functions of RTE cells is reabsorption of the glomerular filtrate, it is not unusual for them to contain substances from the filtrate. RTE cells absorb bilirubin present in the filtrate as the result of liver damage, such as occurs with viral hepatitis, and appear a deep yellow color. As discussed in Chapter 5, hemoglobin present in the filtrate is absorbed by the RTE cells and converted to hemosiderin. Therefore, following episodes of hemoglobinuria (transfusion reactions, paroxysmal nocturnal hemoglobinuria, etc.), the RTE cells may contain the characteristic yellow-brown hemosiderin granules. The granules may also be seen free-floating in the urine sediment. Confirmation of the presence of hemosiderin is performed by staining the urine sediment with Prussian blue. The iron-containing hemosiderin granules stain blue (Fig. 6–32). Oval Fat Bodies RTE cells absorb lipids that are present in the glomerular filtrate. They then appear highly refractile, and the nucleus may be more difficult to observe. These lipid-containing RTE cells are called oval fat bodies (Fig. 6–33). They are usually seen in conjunction with free-floating fat droplets. Identification of oval fat bodies is confirmed by staining the urine sediment with Sudan III or Oil Red O fat stains and examining the sediment using polarized microscopy. The droplets are composed of triglycerides, neutral fats, and cholesterol. Fat stains stain triglycerides and neutral fats, producing orange-red droplets (Fig. 6–34). Examination of the urine sediment using polarized light results in the appearance of characteristic Maltese cross formations in droplets containing cholesterol (Fig. 6–35). Urine sediments negative for fat after staining should still be checked using polarized light in case only cholesterol is present. Likewise, staining should be performed on urine sediments negative under polarized light. Oval fat bodies are reported as the average number per hpf. Free-floating fat droplets also stain or polarize depending on their composition. They may be observed floating on the top of the specimen. Care should be taken not to confuse the droplets with starch and crystal particles that also polarize. Specimen contamination by vaginal preparations and Figure 6–33 Oval fat body (×400). Figure 6–32 Prussian blue–stained hemosiderin granules. Figure 6–34 Sudan III-stained oval fat body (×400). 3920_Ch06_099-146 23/01/14 10:18 AM Page 118 118 Part Two | Urinalysis Brightfield Polarized SUMMARY 6-3 Epithelial Cells Squamous Cells Appearance: Sources of error: Reporting: Figure 6–35 Oval fat body under bright-field (left) and polarized (right) microscopy. Notice the Maltese cross formation (arrow) (×400). lubricants used in specimen collection must be considered when only free-floating fat droplets are present. Lipiduria is most frequently associated with damage to the glomerulus caused by the nephrotic syndrome (see Chapter 7). It is also seen with severe tubular necrosis, diabetes mellitus, and in trauma cases that cause release of bone marrow fat from the long bones. In lipid-storage diseases, large fat-laden histiocytes may also be present. They can be differentiated from oval fat bodies by their large size. In cases of acute tubular necrosis, RTE cells containing large, nonlipid-filled vacuoles may be seen along with normal renal tubular cells and oval fat bodies. Referred to as “bubble cells,” they appear to represent injured cells in which the endoplasmic reticulum has dilated prior to cell death.18 Complete urinalysis correlations: Largest cells in the sediment with abundant, irregular cytoplasm and prominent nuclei Rarely encountered, folded cells may resemble casts Rare, few, moderate, or many per lpf Clarity Transitional Cells Appearance: Sources of error: Reporting: Complete urinalysis correlations: Spherical, polyhedral, or caudate with centrally located nucleus Spherical forms resemble RTE cells Rare, few, moderate, or many per hpf Clarity Blood, if malignancyassociated RTE Cells Appearance: Bacteria Sources of error: Bacteria are not normally present in urine. However, unless specimens are collected under sterile conditions (catheterization), a few bacteria are usually present as a result of vaginal, urethral, external genitalia, or collection-container contamination. These contaminant bacteria multiply rapidly in specimens that remain at room temperature for extended periods, but are of no clinical significance. They may produce a positive nitrite test result and also frequently result in a pH above 8, indicating an unacceptable specimen. Bacteria may be present in the form of cocci (spherical) or bacilli (rods). Owing to their small size, they must be observed and reported using high-power magnification. They are reported as few, moderate, or many per high-power field. To be considered significant for UTI, bacteria should be accompanied by WBCs. Some laboratories report bacteria only when observed in fresh specimens in conjunction with WBCs (Fig. 6–36 A and B). The presence of motile organisms in a drop of fresh urine collected under sterile conditions correlates well with a positive urine culture. Observing bacteria for motility also is useful in differentiating them from similarly appearing amorphous phosphates and urates. The use of phase microscopy aids in the visualization of bacteria. Reporting: Complete urinalysis correlations: Rectangular, columnar, round, oval or, cuboidal with an eccentric nucleus possibly bilirubinstained or hemosiderin-laden Spherical transitional cells Granular casts Average number per 10 hpfs Leukocyte esterase and nitrite (pyelonephritis) Color Clarity Protein Bilirubin (hepatitis) Blood Oval Fat Bodies Appearance: Sources of error: Reporting: Complete urinalysis correlations: Highly refractile RTE cells Confirm with fat stains and polarized microscopy Average number per hpf Clarity Blood Protein Free fat droplets/fatty casts 3920_Ch06_099-146 23/01/14 10:18 AM Page 119 Chapter 6 | Microscopic Examination of Urine 119 A B A B Figure 6–36 A. Rod-shaped bacteria often seen in urinary tract infections. B. KOVA-stained bacteria and WBC (×400). Figure 6–37 A. Budding yeast B. Yeast showing mycelial forms (×400). The presence of bacteria can be indicative of either lower or upper UTI. Specimens containing increased bacteria and leukocytes are routinely followed up with a specimen for quantitative urine culture. The bacteria most frequently associated with UTI are the Enterobacteriaceae (referred to as gramnegative rods); however, the cocci-shaped Staphylococcus and Enterococcus are also capable of causing UTI. The actual bacteria producing an UTI cannot be identified with the microscopic examination. the growth of yeast. As with bacteria, a small amount of yeast entering a specimen as a contaminant multiplies rapidly if the specimen is not examined while fresh. A true yeast infection should be accompanied by the presence of WBCs. Yeast Yeast cells appear in the urine as small, refractile oval structures that may or may not contain a bud. In severe infections, they may appear as branched, mycelial forms (Fig. 6–37 A and B). Yeast cells are reported as rare, few, moderate, or many per hpf. Differentiation between yeast cells and RBCs can sometimes be difficult. Careful observation for budding yeast cells should be helpful, as shown in Figure 6–10. Yeast cells, primarily Candida albicans, are seen in the urine of diabetic patients, immunocompromised patients, and women with vaginal moniliasis. The acidic, glucose-containing urine of patients with diabetes provides an ideal medium for Parasites The most frequent parasite encountered in the urine is Trichomonas vaginalis. The Trichomonas trophozoite is a pearshaped flagellate with an undulating membrane. It is easily identified in wet preparations of the urine sediment by its rapid darting movement in the microscopic field. Trichomonas is usually reported as rare, few, moderate, or many per hpf. When not moving, Trichomonas is more difficult to identify and may resemble a WBC, transitional, or RTE cell. Use of phase microscopy may enhance visualization of the flagella or undulating membrane. T. vaginalis is a sexually transmitted pathogen associated primarily with vaginal inflammation. Infection of the male urethra and prostate is asymptomatic. Males are often asymptomatic carriers (Fig. 6–38). The ova of the bladder parasite Schistosoma haematobium will appear in the urine. However, this parasite is seldom seen 3920_Ch06_099-146 23/01/14 10:19 AM Page 120 120 Part Two | Urinalysis Figure 6–38 Trichomonas vaginalis. Notice the flagella and undulating membrane. (From Leventhal and Cheadle, Ed 6, p 87). A in the United States. It has been associated with bladder cancer in other countries (Fig. 6–39). Fecal contamination of a urine specimen can also result in the presence of ova from intestinal parasites in the urine sediment. The most common contaminant is ova from the pinworm Enterobius vermicularis (Fig. 6–40 A and B). Spermatozoa Spermatozoa are easily identified in the urine sediment by their oval, slightly tapered heads and long, flagella-like tails (Fig. 6–41). Urine is toxic to spermatozoa; therefore, they rarely exhibit the motility observed when examining a semen specimen. Spermatozoa are occasionally found in the urine of both men and women following sexual intercourse, masturbation, or nocturnal emission. They are rarely of clinical significance except in cases of male infertility or retrograde ejaculation in which sperm is expelled into the bladder instead of the urethra. A positive reagent strip test for protein may be seen when increased amounts of semen are present. Laboratory protocols vary with regard to reporting or not reporting the presence of spermatozoa in a urine specimen. Laboratories not reporting its presence cite the lack of clinical significance and possible legal consequences. Laboratories supporting the reporting of spermatozoa cite the possible clinical significance and the minimal possibility of legal consequences.19 B Figure 6–40 A. Enterobius vermicularis ova (×100) B. Enterobius vermicularis ova (×400). Figure 6–41 Spermatozoa (×400). Mucus Figure 6–39 Schistosoma haematobium ova (×300). Eggs are often contained in the last few drops of urine expelled from the bladder. Mucus is a protein material produced by the glands and epithelial cells of the lower genitourinary tract and the RTE cells. Immunologic analysis has shown that uromodulin is a major constituent of mucus. Uromodulin is a glycoprotein excreted by the RTE cells of the distal convoluted tubules and upper collecting ducts. Mucus appears microscopically as thread-like structures with a low refractive index. Subdued light is required when using bright-field microscopy. Care must be taken not to confuse clumps of mucus with hyaline casts. The differentiation 3920_Ch06_099-146 23/01/14 10:19 AM Page 121 Chapter 6 | Microscopic Examination of Urine 121 can usually be made by observing the irregular appearance of the mucous threads (Fig. 6–42 A and B). Mucus threads are reported as rare, few, moderate, or many per lpf. Mucus is more frequently present in female urine specimens. It has no clinical significance when present in either female or male urine. Casts Casts are the only elements found in the urinary sediment that are unique to the kidney. They are formed within the lumens of the distal convoluted tubules and collecting ducts, providing a microscopic view of conditions within the nephron. Their shape is representative of the tubular lumen, with parallel sides and somewhat rounded ends, and they may contain additional elements present in the filtrate. Examination of the sediment for the detection of casts is performed using lower power magnification. When the glass cover-slip method is used, low-power scanning should be performed along the edges of the cover slip. Observation under subdued light is essential, because the cast matrix has a low refractive index. Similar to many other sediment constituents, SUMMARY 6-4 Miscellaneous Structures Bacteria Appearance: Sources of error: Reporting: Complete urinalysis correlations: Small spherical and rod-shaped structures Amorphous phosphates and urates Few, moderate, or many per hpf, the presence of WBCs may be required pH Nitrite LE WBCs Yeast Appearance: Sources of error: Reporting: Complete urinalysis correlations: Small, oval, refractile structures with buds and/or mycelia RBCs Rare, few, moderate, or many per hpf, the presence of WBCs may be required Glucose LE WBCs Trichomonas Appearance: Sources of error: Reporting: Complete urinalysis correlations: A Pear-shaped, motile, flagellated WBCs, renal tubular epithelial cells Rare, few, moderate, or many per hpf LE WBCs Spermatozoa Appearance: Sources of error: Reporting: Complete urinalysis correlations: Tapered oval head with long, thin tail None Present, based on laboratory protocol Protein Mucus Appearance: Sources of error: Reporting: B Figure 6–42 A. Mucus threads (×400). B. Mucus clump (×400). Complete urinalysis correlations: Single or clumped threads with a low refractive index Hyaline casts Rare, few, moderate, or many per lpf None 3920_Ch06_099-146 23/01/14 10:19 AM Page 122 122 Part Two | Urinalysis the cast matrix dissolves quickly in dilute, alkaline urine. Once detected, casts must be further identified as to composition using high-power magnification. They are reported as the average number per 10 lpfs. clinical conditions and will be discussed separately in this section. Cast Composition and Formation The most frequently seen cast is the hyaline type, which consists almost entirely of uromodulin. The presence of zero to two hyaline casts per lpf is considered normal, as is the finding of increased numbers following strenuous exercise, dehydration, heat exposure, and emotional stress.15 Pathologically, hyaline casts are increased in acute glomerulonephritis, pyelonephritis, chronic renal disease, and congestive heart failure. Hyaline casts appear colorless in unstained sediments and have a refractive index similar to that of urine; thus, they can easily be overlooked if specimens are not examined under subdued light (Figs. 6–43 and 6–44). Sternheimer-Malbin stain produces a pink color in hyaline casts. Increased visualization can be obtained by phase microscopy (Fig. 6–45 A and B). The morphology of hyaline casts is varied, consisting of normal parallel sides and rounded ends, cylindroid forms, and wrinkled or convoluted shapes that indicate aging of the cast matrix (Fig. 6–46). The presence of an occasional adhering cell or granule may also be observed (Fig. 6–47) but does not change the cast classification. The major constituent of casts is uromodulin. Other proteins present in the urinary filtrate, such as albumin and immunoglobulins, are also incorporated into the cast matrix. Under normal conditions, uromodulin is excreted at a relatively constant rate. The rate of excretion appears to increase under conditions of stress and exercise, which may account for the transient appearance of hyaline casts when these conditions are present. The protein gels more readily under conditions of urine-flow stasis, acidity, and the presence of sodium and calcium. The extent of protein glycosylation is also important.20 Uromodulin protein is found in both normal and abnormal urine and, as discussed previously, is a major constituent of mucus. It is not detected by reagent strip protein methods. Therefore, the increased urinary protein frequently associated with the presence of casts is caused by underlying renal conditions. Scanning electron microscope studies have provided a step-by-step analysis of the formation of the uromodulin protein matrix21: 1. Aggregation of uromodulin protein into individual protein fibrils attached to the RTE cells 2. Interweaving of protein fibrils to form a loose fibrillar network (urinary constituents may become enmeshed in the network at this time) 3. Further protein fibril interweaving to form a solid structure 4. Possible attachment of urinary constituents to the solid matrix 5. Detachment of protein fibrils from the epithelial cells 6. Excretion of the cast As the cast forms, urinary flow within the tubule decreases as the lumen becomes blocked. The accompanying dehydration of the protein fibrils and internal tension may account for the wrinkled and convoluted appearance of older hyaline casts.22 The width of the cast depends on the size of the tubule in which it is formed. Broad casts may result from tubular distension or, in the case of extreme urine stasis, from formation in the collecting ducts. Formation of casts at the junction of the ascending loop of Henle and the distal convoluted tubule may produce structures with a tapered end. These have been referred to as cylindroids, but they have the same significance as casts. In fact, the presence of urinary casts is termed cylindruria. The appearance of a cast is also influenced by the materials present in the filtrate at the time of its formation and the length of time it remains in the tubule. Any elements present in the tubular filtrate, including cells, bacteria, granules, pigments, and crystals, may become embedded in or attached to the cast matrix. The types of casts found in the sediment represent different Hyaline Casts Figure 6–43 Hyaline casts under low power (×100). A B Figure 6–44 Hyaline cast (A) and amorphous urates (B) attached to mucus pseudocast (×100). 3920_Ch06_099-146 23/01/14 10:19 AM Page 123 Chapter 6 | Microscopic Examination of Urine 123 A Figure 6–47 Hyaline cast containing occasional granules (×400). casts have also been observed in healthy individuals following participation in strenuous contact sports.15 RBC casts are easily detected under low power by their orange-red color. They are more fragile than other casts and may exist as fragments or have a more irregular shape as the result of tightly packed cells adhering to the protein matrix (Figs. 6–48 and 6–49). Examination under high-power magnification should concentrate on determining that a cast matrix is present, thereby differentiating the structure from a B Figure 6–45 A. Hyaline cast (×400). B. Hyaline cast under phase microscopy (×400). Figure 6–48 RBC cast (×400). Figure 6–46 Convoluted hyaline cast (×400). RBC Casts Whereas the finding of RBCs in the urine indicates bleeding from an area within the genitourinary tract, the presence of RBC casts is much more specific, showing bleeding within the nephron. RBC casts are primarily associated with damage to the glomerulus (glomerulonephritis) that allows passage of the cells through the glomerular membrane; however, any damage to the nephron capillary structure can cause their formation. RBC casts associated with glomerular damage are usually associated with proteinuria and dysmorphic erythrocytes. RBC Figure 6–49 KOVA-stained RBC cast under phase microscopy (×400). 3920_Ch06_099-146 23/01/14 10:19 AM Page 124 Part Two | Urinalysis 124 clump of RBCs. Because of the serious diagnostic implications of RBC casts, the actual presence of RBCs must also be verified to prevent the inaccurate reporting of nonexistent RBC casts. It is highly improbable that RBC casts will be present in the absence of free-standing RBCs and a positive reagent strip test for blood (Fig. 6–50). As an RBC cast ages, cell lysis begins and the cast develops a more homogenous appearance, but retains the characteristic orange-red color from the released hemoglobin (Fig. 6–51). These casts may be distinguished as blood casts, indicating greater stasis of urine flow. However, because all casts containing blood have the same clinical significance, this is not considered necessary. Both types of casts are reported as the number of RBC casts per lpf. In the presence of massive hemoglobinuria or myoglobinuria, homogenous orange-red or red-brown casts may be observed. Granular, dirty, brown casts representing hemoglobin degradation products such as methemoglobin may also be present (Fig. 6–52). They are associated with the acute tubular necrosis often caused by the toxic effects of massive hemoglobinuria that can lead to renal failure. These dirty, brown casts must be present in conjunction with other pathologic findings such as RTE cells and a positive reagent strip test for blood. Figure 6–52 Granular, dirty, brown cast (×400). WBC Casts The appearance of WBC casts in the urine signifies infection or inflammation within the nephron. They are most frequently associated with pyelonephritis and are a primary marker for distinguishing pyelonephritis (upper UTI) from cystitis (lower UTI). However, they are also present in nonbacterial inflammations such as acute interstitial nephritis and may accompany RBC casts in glomerulonephritis. WBC casts are visible under low-power magnification but must be positively identified using high power. Most frequently, WBC casts are composed of neutrophils; therefore, they may appear granular, and, unless disintegration has occurred, multilobed nuclei will be present (Fig. 6–53). Supravital staining may be necessary to demonstrate the characteristic nuclei (Fig. 6–54). It is particularly helpful for differentiating WBC casts from RTE casts. Observation of free WBCs in the sediment is also essential (Fig. 6–55). Bacteria are present in cases of pyelonephritis, but are not present with acute interstitial nephritis; however, eosinophil casts may be present in appropriately stained specimens (Hansel and Wright’s stains). Figure 6–50 Disintegrating RBC cast. Notice the presence of free RBCs (arrows) to confirm identification. A B Figure 6–51 Cast containing hemoglobin pigment. A comparison of RBCs (A) and yeast (B) also can be made (×400). Figure 6–53 WBC cast. Notice the free WBCs to aid in identification. 3920_Ch06_099-146 23/01/14 10:19 AM Page 125 Chapter 6 | Microscopic Examination of Urine 125 Identification of bacterial casts can be difficult, because packed casts packed with bacteria can resemble granular casts. Their presence should be considered when WBC casts and many free WBCs and bacteria are seen in the sediment. Confirmation of bacterial casts is best made by performing a Gram stain on the dried or cytocentrifuged sediment. Epithelial Cell Casts Figure 6–54 KOVA-stained WBC cast (×400). Figure 6–55 Disintegrating WBC cast (×400). Casts tightly packed with WBCs may have irregular borders. These structures should be carefully examined to determine that a cast matrix is present. WBCs frequently form clumps, and these do not have the same significance as casts (Fig. 6–56). Bacterial Casts Bacterial casts containing bacilli both within and bound to the protein matrix are seen in pyelonephritis.23 They may be pure bacterial casts or mixed with WBCs. Figure 6–56 WBC clump. Notice the absence of a cast matrix. Casts containing RTE cells represent the presence of advanced tubular destruction, producing urinary stasis along with disruption of the tubular linings. Similar to RTE cells, they are associated with heavy metal and chemical or drug-induced toxicity, viral infections, and allograft rejection. They also accompany WBC casts in cases of pyelonephritis. As discussed previously, the fibrils of uromodulin protein that make up the cast matrix remain attached to the RTE cells that produce them; therefore, the observation of an occasional tubular cell attached to a hyaline cast can be expected. When tubular damage is present, some cells may be incorporated into the cast matrix, but the majority will be very noticeably attached to the cast surface. Owing to the formation of casts in the distal convoluted tubule, the cells visible on the cast matrix are the smaller, round, and oval cells (Fig. 6–57). They may be difficult to differentiate from WBCs, particularly if degeneration has occurred. Staining and the use of phase microscopy can be helpful to enhance the nuclear detail needed for identification (Figs. 6–58 A and B and 6–59). Fragments of epithelial tissue may also be attached to the cast matrix. Bilirubin-stained RTE cells are seen in cases of hepatitis (see Fig. 6–59). Fatty Casts Fatty casts are seen in conjunction with oval fat bodies and free fat droplets in disorders causing lipiduria. They are most frequently associated with the nephrotic syndrome, but are also seen in toxic tubular necrosis, diabetes mellitus, and crush injuries. Fatty casts are highly refractile under bright-field microscopy. The cast matrix may contain few or many fat droplets, and intact oval fat bodies may be attached to the Figure 6–57 RTE cell cast (×400). 3920_Ch06_099-146 23/01/14 10:19 AM Page 126 126 Part Two | Urinalysis A Figure 6–60 Fatty cast showing adherence of fat droplets (arrows) to cast matrix (×400). B Figure 6–58 A. KOVA-stained RTE cell cast (×400). B. KOVA-stained RTE cell cast under phase microscopy (×400). Figure 6–61 Fatty cast (×400). Figure 6–59 RTE cast with bilirubin-stained cells (×400). matrix (Figs. 6–60, 6–61, 6–62). Confirmation of fatty casts is performed using polarized microscopy and Sudan III or Oil Red O fat stains. As discussed previously, cholesterol demonstrates characteristic Maltese cross formations under polarized light, and triglycerides and neutral fats stain orange with fat stains. Fats do not stain with Sternheimer-Malbin stains. Mixed Cellular Casts Considering that a variety of cells may be present in the urinary filtrate, observing casts containing multiple cell types is not uncommon. Mixed cellular casts most frequently Figure 6–62 Fatty cast under phase microscopy (×400). encountered include RBC and WBC casts in glomerulonephritis and WBC and RTE cell casts, or WBC and bacterial casts in pyelonephritis. The presence of mixed elements in a cast may make identification more difficult. Staining or phase microscopy aids in the identification. When mixed casts are present, there should also be homogenous casts of at least one of the cell types, and they will be the primary diagnostic marker. For example, in 3920_Ch06_099-146 23/01/14 10:19 AM Page 127 Chapter 6 | Microscopic Examination of Urine 127 glomerulonephritis, the predominant casts will be RBC, and in pyelonephritis, the predominant casts will be WBC. Bacteria are often incorporated into WBC casts and provide little additional diagnostic significance. Laboratory protocol should be followed in the reporting of mixed cellular casts. Granular Casts Coarsely and finely granular casts are frequently seen in the urinary sediment and may be of pathologic or nonpathologic significance. It is not considered necessary to distinguish between coarsely and finely granular casts. The origin of the granules in nonpathologic conditions appears to be from the lysosomes excreted by RTE cells during normal metabolism.24 It is not unusual to see hyaline casts containing one or two of these granules. Increased cellular metabolism occurring during periods of strenuous exercise accounts for the transient increase of granular casts that accompany the increased hyaline casts (Figs. 6–63 and 6–64).15 In disease states, granules may represent disintegration of cellular casts and tubule cells or protein aggregates filtered by the glomerulus (Figs. 6–65 and 6–66). Scanning electron microscope studies have confirmed that granular casts seen in conjunction with WBC casts contain WBC granules of varying sizes.25 Urinary stasis allowing the casts to remain in the Figure 6–65 Granular disintegrating cellular cast (×400). B C A B Figure 6–66 Coarsely granular cast (A), squamous epithelial cell (B), and mucus (C) (×400). A Figure 6–63 Finely granular cast (A) and uric acid crystals (B) (×400). tubules must be present for granules to result from disintegration of cellular casts. Granular casts occurring as a result of cellular disintegration may contain an occasional recognizable cell. Granular casts are easily visualized under low-power microscopy. However, final identification should be performed using high power to determine the presence of a cast matrix. Artifacts, such as clumps of small crystals and fecal debris, may occur in shapes resembling casts and must be differentiated. As mentioned previously, columnar RTE cells may also resemble granular casts, and staining for nuclear detail may be required. When granular casts remain in the tubules for extended periods, the granules further disintegrate, and the cast matrix develops a waxy appearance. The structure becomes more rigid, the ends of the casts may appear jagged or broken, and the diameter becomes broader (Fig. 6–67). Waxy Casts Figure 6–64 Granular cast formed at a tubular bend (×400). Waxy casts are representative of extreme urine stasis, indicating chronic renal failure. They are usually seen in conjunction with other types of casts associated with the condition that has caused the renal failure. 3920_Ch06_099-146 23/01/14 10:19 AM Page 128 128 Part Two | Urinalysis Figure 6–67 Granular cast degenerating into waxy cast (×400). Figure 6–70 KOVA-stained waxy cast (×400). The brittle, highly refractive cast matrix from which these casts derive their name is believed to be caused by degeneration of the hyaline cast matrix and any cellular elements or granules contained in the matrix.22,24 Waxy casts are more easily visualized than hyaline casts because of their higher refractive index. As a result of the brittle consistency of the cast matrix, they often appear fragmented with jagged ends and have notches in their sides (Figs. 6–68 and 6–69). With supravital stains, waxy casts stain a homogenous, dark pink (Fig. 6–70). Broad Casts Often referred to as renal failure casts, broad casts like waxy casts represent extreme urine stasis. As a mold of the distal convoluted tubules, the presence of broad casts indicates destruction (widening) of the tubular walls. Also, when the flow of urine to the larger collecting ducts becomes severely compromised, casts form in this area and appear broad. All types of casts may occur in the broad form. However, considering the accompanying urinary stasis, the most commonly seen broad casts are granular and waxy (Figs. 6–71 and 6–72). Bile-stained broad, waxy casts are seen as the result of the tubular necrosis caused by viral hepatitis (Fig. 6–73). Urinary Crystals Figure 6–68 KOVA-stained waxy casts (×100). Figure 6–69 KOVA-stained waxy casts (×200). Crystals frequently found in the urine are rarely of clinical significance. They may appear as true geometrically formed structures or as amorphous material. The primary reason for the identification of urinary crystals is to detect the presence of the relatively few abnormal types that may represent such disorders as liver disease, inborn errors of metabolism, or renal damage caused by crystallization of medications compounds within the tubules. Crystals are usually reported as rare, few, moderate, or many per hpf. Abnormal crystals may be averaged and reported per lpf. Figure 6–71 KOVA-stained broad waxy cast (×400). 3920_Ch06_099-146 23/01/14 10:19 AM Page 129 Chapter 6 | Microscopic Examination of Urine 129 Crystal Formation Figure 6–72 Broad granular cast becoming waxy (×400). Crystals are formed by the precipitation of urine solutes, including inorganic salts, organic compounds, and medications (iatrogenic compounds). Precipitation is subject to changes in temperature, solute concentration, and pH, which affect solubility. Solutes precipitate more readily at low temperatures. Therefore, the majority of crystal formation takes place in specimens that have remained at room temperature or been refrigerated prior to testing. Crystals are extremely abundant in refrigerated specimens and often present problems because they obscure clinically significant sediment constituents. As the concentration of urinary solutes increases, their ability to remain in solution decreases, resulting in crystal formation. The presence of crystals in freshly voided urine is most frequently associated with concentrated (high specific gravity) specimens. A valuable aid in the identification of crystals is the pH of the specimen because this determines the type of chemicals precipitated. In general, organic and iatrogenic compounds crystallize more easily in an acidic pH, whereas inorganic salts are less soluble in neutral and alkaline solutions. An exception is calcium oxalate, which precipitates in both acidic and neutral urine. General Identification Techniques Figure 6–73 Broad bile-stained waxy cast (×400). SUMMARY 6-5 Urine Casts Hyaline Appearance: Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: The most commonly seen crystals have very characteristic shapes and colors; however, variations do occur and can present identification problems, particularly when they resemble abnormal crystals. As discussed previously, the first consideration when identifying crystals is the urine pH. In fact, crystals are routinely classified not only as normal and abnormal, but also Colorless, homogenous matrix Mucus, fibers, hair, increased lighting Average number per lpf Protein Blood (exercise) Color (exercise) Glomerulonephritis Pyelonephritis Chronic renal disease Congestive heart failure Stress and exercise Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: WBC Appearance: Sources of error: Reporting: Complete urinalysis correlations: RBC Appearance: Orange-red color, cast matrix containing RBCs RBC clumps Average number per lpf RBCs Blood Protein Glomerulonephritis Strenuous exercise Clinical significance: Cast matrix containing WBCs WBC clumps Average number per lpf WBCs Protein LE Pyelonephritis Acute interstitial nephritis Continued 3920_Ch06_099-146 23/01/14 10:19 AM Page 130 130 Part Two | Urinalysis SUMMARY 6-5 Urine Casts—cont’d Bacterial Appearance: Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: Bacilli bound to protein matrix Granular casts Average number per lpf WBC casts (pyelonephritis) WBCs LE Nitrite Protein Bacteria Pyelonephritis Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: Fatty Appearance: Epithelial Cell Appearance: Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: RTE cells attached to protein matrix WBC cast Average number per lpf Protein RTE cells Renal tubular damage Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: Granular Appearance: Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: Coarse and fine granules in a cast matrix Clumps of small crystals Columnar RTE cells Average number per lpf Protein Cellular casts RBCs WBCs Glomerulonephritis Pyelonephritis Stress and exercise Waxy Appearance: Fibers and fecal material Average number per lpf Protein Cellular casts Granular casts WBCs RBCs Stasis of urine flow Chronic renal failure Fat droplets and oval fat bodies attached to protein matrix Fecal debris Average number per lpf Protein Free fat droplets Oval fat bodies Nephrotic syndrome Toxic tubular necrosis Diabetes mellitus Crush injuries Broad Appearance: Sources of error: Reporting: Complete urinalysis correlations: Clinical significance: Wider than normal cast matrix Fecal material, fibers Average number per lpf Protein WBCs RBCs Granular casts Waxy casts Extreme urine stasis Renal failure Highly refractile cast with jagged ends and notches as to their appearance in acidic or alkaline urine. All abnormal crystals are found in acidic urine. Additional aids in crystal identification include the use of polarized microscopy and solubility characteristics of the crystals. The geometric shape of a crystal determines its birefringence and, therefore, its ability to polarize light. Although the size of a particular crystal may vary (slower crystallization produces larger crystals), the basic structure remains the same. Therefore, polarization characteristics for a particular crystal are constant for identification purposes. Just as changes in temperature and pH contribute to crystal formation, reversal of these changes can cause crystals to dissolve. These solubility characteristics can be used to aid in identification. Amorphous urates that frequently form in refrigerated specimens and obscure sediments may dissolve if the specimen is warmed. Amorphous phosphates require acetic acid to dissolve, and this is not practical, as formed elements, such as RBCs, will also be destroyed. When solubility characteristics are needed for identification, the sediment should be aliquoted to 3920_Ch06_099-146 23/01/14 10:19 AM Page 131 Chapter 6 | Microscopic Examination of Urine 131 prevent destruction of other elements. In Table 6–6, characteristics for the most commonly encountered crystals are provided. Normal Crystals Seen in Acidic Urine The most common crystals seen in acidic urine are urates, consisting of amorphous urates, uric acid, acid urates, and sodium urates. Microscopically, most urate crystals appear yellow to reddish brown and are the only normal crystals found in acidic urine that appear colored. Table 6–6 Amorphous urates appear microscopically as yellowbrown granules (Fig. 6–74). They may occur in clumps resembling granular casts and attached to other sediment structures (Fig.6–75). Amorphous urates are frequently encountered in specimens that have been refrigerated and produce a very characteristic pink sediment. Accumulation of the pigment, uroerythrin, on the surface of the granules is the cause of the pink color. Amorphous urates are found in acidic urine with a pH greater than 5.5, whereas uric acid crystals can appear when the pH is lower. Major Characteristics of Normal Urinary Crystals Crystal pH Color Uric acid Acid Yellow-brown (rosettes, wedges) Amorphous urates Acid Brick dust or yellow brown Calcium oxalate Acid/neutral (alkaline) Colorless (envelopes, oval, dumbbell) Amorphous phosphates Alkaline/ neutral White–colorless Calcium phosphate Alkaline/ neutral Colorless Triple phosphate Alkaline Colorless (“coffin lids”) Ammonium biurate Alkaline Yellow-brown (“thorny apples”) Calcium carbonate Alkaline Colorless (dumbbells) Appearance 3920_Ch06_099-146 23/01/14 10:19 AM Page 132 132 Part Two | Urinalysis Figure 6–74 Amorphous urates (×400). Figure 6–77 Clump of uric acid crystals (×400). Notice the whetstone, not hexagonal, shape that differentiates uric acid crystals from cystine crystals. cystine crystals (Fig. 6–78 A and B). Increased amounts of uric acid crystals, particularly in fresh urine, are associated with increased levels of purines and nucleic acids and are seen in patients with leukemia who are receiving chemotherapy, in patients with Lesch-Nyhan syndrome (see Chapter 8), and sometimes in patients with gout. Acid urates and sodium urates are rarely encountered and, like amorphous urates, are seen in less acidic urine. They are frequently seen in conjunction with amorphous urates and Figure 6–75 Amorphous urates attached to a fiber. Uric acid crystals are seen in a variety of shapes, including rhombic, four-sided flat plates (whetstones), wedges, and rosettes. They usually appear yellow-brown, but may be colorless and have a six-sided shape, similar to cystine crystals (Figs. 6–76 and 6–77). Uric acid crystals are highly birefringent under polarized light, which aids in distinguishing them from A B Figure 6–76 Uric acid crystals (×400). Figure 6–78 A. Uric acid crystals under polarized light (×100). B. Uric acid crystals under polarized light (×400). 3920_Ch06_099-146 23/01/14 10:19 AM Page 133 Chapter 6 | Microscopic Examination of Urine 133 have little clinical significance. Acid urates appear as larger granules and may have spicules similar to the ammonium biurate crystals seen in alkaline urine. Sodium urate crystals are needle-shaped and are seen in synovial fluid during episodes of gout, but may also appear in the urine. Calcium oxalate crystals are frequently seen in acidic urine, but they can be found in neutral urine and even rarely in alkaline urine. The most common form of calcium oxalate crystals is the dihydrate that is easily recognized as a colorless, octahedral envelope or as two pyramids joined at their bases (Figs. 6–79, 6–80, and 6–81). Less characteristic and less frequently seen is the monohydrate form (Fig. 6–82). Monohydrate calcium oxalate crystals are oval or dumbbell shaped. Both the dihydrate and monohydrate forms are birefringent under polarized light. This may be helpful to distinguish the monohydrate form from nonpolarizing RBCs. Calcium oxalate crystals are sometimes seen in clumps attached to mucous strands and may resemble casts. The finding of clumps of calcium oxalate crystals in fresh urine may be related to the formation of renal calculi, because the majority of renal calculi are composed of calcium oxalate. They are also associated with foods high in oxalic acid, such Figure 6–81 Attached classic dihydrate calcium oxalate crystals (×400). Figure 6–82 Monohydrate calcium oxalate crystals (×400). Figure 6–79 Classic dihydrate calcium oxalate crystals (×400). as tomatoes and asparagus, and ascorbic acid, because oxalic acid is an end product of ascorbic acid metabolism. The primary pathologic significance of calcium oxalate crystals is the very noticeable presence of the monohydrate form in cases of ethylene glycol (antifreeze) poisoning. The monohydrate form is most frequently seen in children and pets because antifreeze tastes sweet and uncovered containers left in the garage can be very tempting! Massive amounts of crystals are frequently produced in these cases. Normal Crystals Seen in Alkaline Urine Figure 6–80 Classic dihydrate calcium oxalate crystals under phase microscopy (×400). Phosphates represent the majority of the crystals seen in alkaline urine and include amorphous phosphate, triple phosphate, and calcium phosphate. Other normal crystals associated with alkaline urine are calcium carbonate and ammonium biurate. Amorphous phosphates are granular in appearance, similar to amorphous urates (Figs. 6–83 and 6–84). When present in large quantities following specimen refrigeration, they cause a white precipitate that does not dissolve on warming. They can be differentiated from amorphous urates by the color of the sediment and the urine pH. 3920_Ch06_099-146 23/01/14 10:19 AM Page 134 134 Part Two | Urinalysis Figure 6–83 Amorphous phosphates (×400). Urine pH 7.0. Figure 6–86 Triple phosphate crystals (arrow) and amorphous phosphates (×400). Figure 6–87 Calcium carbonate crystals (×400). Figure 6–84 Amorphous phosphates (×400). Triple phosphate (ammonium magnesium phosphate) crystals are commonly seen in alkaline urine. In their routine form, they are easily identified by their prism shape that frequently resembles a “coffin lid” (Figs. 6–85 and 6–86). As they disintegrate, the crystals may develop a feathery appearance. Figure 6–85 Triple phosphate crystal (×400). Triple phosphate crystals are birefringent under polarized light. They have no clinical significance; however, they are often seen in highly alkaline urine associated with the presence of ureasplitting bacteria. Calcium phosphate crystals are not frequently encountered. They may appear as colorless, flat rectangular plates or thin prisms often in rosette formations. The rosette forms may be confused with sulfonamide crystals when the urine pH is in the neutral range. Calcium phosphate crystals dissolve in dilute acetic acid and sulfonamides do not. They have no clinical significance, although calcium phosphate is a common constituent of renal calculi. Calcium carbonate crystals are small and colorless, with dumbbell or spherical shapes (Fig. 6–87). They may occur in clumps that resemble amorphous material, but they can be distinguished by the formation of gas after the addition of acetic acid. They are also birefringent, which differentiates them from bacteria. Calcium carbonate crystals have no clinical significance. Ammonium biurate crystals exhibit the characteristic yellow-brown color of the urate crystals seen in acidic urine. They are frequently described as “thorny apples” because of their appearance as spicule-covered spheres (Fig. 6–88). Except for their occurrence in alkaline urine, ammonium biurate crystals resemble other urates in that they dissolve at 60°C and 3920_Ch06_099-146 23/01/14 10:19 AM Page 135 Chapter 6 | Microscopic Examination of Urine 135 Figure 6–88 Ammonium biurate crystals (×400). Notice the “thorny apple” appearance. (Courtesy of Kenneth L. McCoy, MD.) convert to uric acid crystals when glacial acetic acid is added. Ammonium biurate crystals are almost always encountered in old specimens and may be associated with the presence of the ammonia produced by urea-splitting bacteria (Figs. 6–89 A and B and 6–90). Figure 6–90 Ammonium biurate crystals (×400). Note thorns (arrow). Abnormal Urine Crystals Abnormal urine crystals are found in acidic urine or rarely in neutral urine. Most abnormal crystals have very characteristic shapes. However, their identity can be confirmed by patient information, including disorders and medication (Table 6–7). Iatrogenic crystals can be caused by a variety of compounds, particularly when they are administered in high concentrations. They may be of clinical significance when they precipitate in the renal tubules. The most commonly encountered iatrogenic crystals are discussed in this section. A B Figure 6–89 Ammonium biurate crystals A. Ammonium biurate and triple phosphate crystals (×100). Note thorn (arrow). B. Ammonium biurate and triple phosphate crystals (×400). Cystine Crystals Cystine crystals are found in the urine of persons who inherit a metabolic disorder that prevents reabsorption of cystine by the renal tubules (cystinuria). Persons with cystinuria have a tendency to form renal calculi, particularly at an early age. Cystine crystals appear as colorless, hexagonal plates and may be thick or thin (Figs. 6–91 and 6–92). Disintegrating forms may be seen in the presence of ammonia. They may be difficult to differentiate from colorless uric acid crystals. Uric acid crystals are very birefringent under polarized microscopy, whereas only thick cystine crystals have polarizing capability. Positive confirmation of cystine crystals is made using the cyanide-nitroprusside test (see Chapter 8). Cholesterol Crystals Cholesterol crystals are rarely seen unless specimens have been refrigerated, because the lipids remain in droplet form. However, when observed, they have a most characteristic appearance, resembling a rectangular plate with a notch in one or more corners (Fig. 6–93). They are associated with disorders producing lipiduria, such as the nephrotic syndrome, and are seen in conjunction with fatty casts and oval fat bodies. Cholesterol crystals are highly birefringent with polarized light (Fig. 6–94). 3920_Ch06_099-146 23/01/14 10:19 AM Page 136 136 Part Two | Urinalysis Table 6–7 Major Characteristics of Abnormal Urinary Crystals Crystal pH Color/Form Disorders Cystine Acid Colorless (hexagonal plates) Inherited cystinuria Cholesterol Acid Colorless (notched plates) Nephrotic syndrome Leucine Acid/neutral Yellow (concentric circles) Liver disease Tyrosine Acid/neutral Colorless–yellow (needles) Liver disease Bilirubin Acid Yellow Liver disease Sulfonamides Acid/neutral Varied Infection treatment Radiographic dye Acid Colorless (flat plates) Radiographic procedure Ampicillin Acid/neutral Colorless (needles) Infection treatment Appearance Radiographic Dye Crystals Crystals of radiographic contrast media have a very similar appearance to cholesterol crystals and also are highly birefringent. Differentiation is best made by comparison of the other urinalysis results and the patient history. As mentioned previously, cholesterol crystals should be accompanied by other lipid elements and heavy proteinuria. Likewise, the specific gravity of a specimen containing radiographic contrast media is markedly elevated when measured by refractometer. Figure 6–91 Cystine crystals (×400). Crystals Associated With Liver Disorders In the presence of severe liver disorders, three rarely seen crystals may be found in the urine sediment. They are crystals of tyrosine, leucine, and bilirubin. 3920_Ch06_099-146 23/01/14 10:19 AM Page 137 Chapter 6 | Microscopic Examination of Urine 137 Figure 6–92 Clump of cystine crystals (×400). Notice the hexagonal shape still visible. Figure 6–93 Cholesterol crystals. Notice the notched corners (×400). Figure 6–95 Tyrosine crystals in fine needle clumps (×400). Figure 6–96 Tyrosine crystals in rosette forms (×400). Figure 6–97 Leucine crystals (×400). Notice the concentric circles. Figure 6–94 Cholesterol crystals under polarized light (×400). Tyrosine crystals appear as fine colorless to yellow needles that frequently form clumps or rosettes (Figs. 6–95 and 6–96). They are usually seen in conjunction with leucine crystals in specimens with positive chemical test results for bilirubin. Tyrosine crystals may also be encountered in inherited disorders of amino acid metabolism (see Chapter 8). Leucine crystals are yellow-brown spheres that demonstrate concentric circles and radial striations (Fig. 6–97). They are seen less frequently than tyrosine crystals and, when present, should be accompanied by tyrosine crystals. Bilirubin crystals are present in hepatic disorders producing large amounts of bilirubin in the urine. They appear as clumped needles or granules with the characteristic yellow color of bilirubin (Fig. 6–98). A positive chemical test result for bilirubin would be expected. In disorders that produce renal tubular damage, such as viral hepatitis, bilirubin crystals may be found incorporated into the matrix of casts. 3920_Ch06_099-146 23/01/14 10:19 AM Page 138 138 Part Two | Urinalysis Figure 6–98 Bilirubin crystals. Notice the classic bright yellow color (×400). Sulfonamide Crystals Prior to the development of more soluble sulfonamides, the finding of these crystals in the urine of patients being treated for UTIs was common. Inadequate patient hydration was and still is the primary cause of sulfonamide crystallization. The appearance of sulfonamide crystals in fresh urine can suggest the possibility of tubular damage if crystals are forming in the nephron. A variety of sulfonamide medications are currently on the market; therefore, one can expect to encounter a variety of crystal shapes and colors. Shapes most frequently encountered include needles, rhombics, whetstones, sheaves of wheat, and rosettes with colors ranging from colorless to yellow-brown (Figs. 6–99 and 6–100). A check of the patient’s medication history aids in the identification confirmation. Figure 6–100 Sulfa crystals, WBCs, and bacteria seen in UTI (×400). A Ampicillin Crystals Precipitation of antibiotics is not frequently encountered except for the rare observation of ampicillin crystals following massive doses of this penicillin compound without adequate hydration. Ampicillin crystals appear as colorless needles that tend to form bundles following refrigeration (Fig. 6–101 A and B). Knowledge of the patient’s history can aid in the identification. B Figure 6–101 Ampicillin crystals. A. Nonrefrigerated ampicillin crystals. (×400). B. Ampicillin crystals after refrigeration (×400). Urinary Sediment Artifacts Figure 6–99 Sulfa crystals in rosette form (×400). Contaminants of all types can be found in urine, particularly in specimens collected under improper conditions or in dirty containers. The most frequently encountered artifacts include starch, oil droplets, air bubbles, pollen grains, fibers, and fecal contamination. Because artifacts frequently resemble pathologic elements such as RBCs and casts, artifacts can present a major problem to students. They are often very highly refractile or occur in a different microscopic plane than the true sediment constituents. The reporting of artifacts is not necessary. 3920_Ch06_099-146 23/01/14 10:19 AM Page 139 Chapter 6 | Microscopic Examination of Urine 139 Starch granule contamination may occur when cornstarch is the powder used in powdered gloves. The granules are highly refractile spheres, usually with a dimpled center (Fig. 6–102). They resemble fat droplets when polarized, producing a Maltese cross formation. Starch granules may also occasionally be confused with RBCs. Differentiation between starch and pathologic elements can be made by considering other urinalysis results, including chemical tests for blood or protein and the presence of oval fat bodies or fatty casts. Oil droplets and air bubbles also are highly refractile and may resemble RBCs to inexperienced laboratory personnel. Oil droplets may result from contamination by immersion oil or lotions and creams and maybe seen with fecal contamination (Fig. 6–103). Air bubbles occur when the specimen is placed under a cover slip. The presence of these artifacts should be considered in the context of the other urinalysis results. Pollen grains are seasonal contaminants that appear as spheres with a cell wall and occasional concentric circles (Fig. 6–104). Like many artifacts, their large size may cause them to be out of focus with true sediment constituents. Hair and fibers from clothing and diapers may initially be mistaken for casts (Figs. 6–105 and 6–106), though they are usually much longer and more refractile. Examination under polarized light can frequently differentiate between fibers and casts (Fig. 6–107). Fibers often polarize, whereas casts, other than fatty casts, do not polarize. Figure 6–104 Pollen grain. Notice the concentric circles (×400). Figure 6–105 Fiber and squamous epithelial cell (×400). Figure 6–102 Starch granules. Notice the dimpled center (×400). Figure 6–106 Fiber under polarized light (×100). Figure 6–103 Fecal material and oil artifacts (×400). Improperly collected specimens or rarely the presence of a fistula between the intestinal and urinary tracts may produce fecal specimen contamination. Fecal artifacts may appear as plant and meat fibers or as brown amorphous material in a variety of sizes and shapes (Fig. 6–108). 3920_Ch06_099-146 23/01/14 10:19 AM Page 140 140 Part Two | Urinalysis Figure 6–107 Diaper fiber resembling a cast. Notice the refractility (×400). Figure 6–108 Vegetable fiber resembling waxy cast (×400). Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Mynahan, C: Evaluation of macroscopic urinalysis as a screening procedure. Lab Med 15(3):176–179, 1984. 2. Tetrault, GA: Automated reagent strip urinalysis: Utility in reducing work load of urine microscopy and culture. Lab Med 25:162–167, 1994. 3. CLSI. Urinalysis; Approved Guideline, ed 3. CLSI document GP16–A3, Wayne, PA: Clinical and Laboratory Standards Institute. 2009. 4. Schumann, GB, and Tebbs, RD: Comparison of slides used for standardized routine microscopic urinalysis. J Med Technol 3(1):54–58, 1986. 5. Addis, T: The number of formed elements in the urinary sediment of normal individuals. J Clin Invest 2(5):409–415, 1926. 6. Sternheimer, R, and Malbin, R: Clinical recognition of pyelonephritis with a new stain for urinary sediments. Am J Med 11:312–313, 1951. 7. Microscope Techniques—Phase Contrast. Web site: http://www. micro.magnet.fsuj.edu/primer/techniques/phase.html 8. Polarizing and Interference Contrast Microscopy. Web site: http://www.rrz.uni-hamburg.de/biologic/b 9. Olympus Microscopy Resource Center: Specialized Microscopy Techniques: Fluorescence. Web site: http://www.olympusmicro. com/ primer/techniques/fluorescence/fluorhome.html. Accessed December 19, 2006. 10. Simpson, LO: Effects of normal and abnormal urine on red cell shape. Nephron 60(3):383–384, 1992. 11. Stapleton, FB: Morphology of urinary red blood cells: A simple guide in localizing the site of hematuria. Pediatr Clin North Am 34(3):561–569, 1987. 12. Fassett, EG, et al: Urinary red cell morphology during exercise. Am J Clin Pathol 285(6353):1455–1457, 1982. 13. Kohler, H, Wandel, E, and Brunch, B: Acanthocyturia: A characteristic marker for glomerular bleeding. Int Soc Nephrol 40:115–120, 1991. 14. Tomita, M, et al: A new morphological classification of urinary erythrocytes for differential diagnosis of hematuria. Clin Nephrol 37(2):84–89, 1992. 15. Haber, MH, Lindner, LE, and Ciofalo, LN: Urinary casts after stress. Lab Med 10(6):351–355, 1979. 16. Corwin, HL, Bray, RA, and Haber, MH: The detection and interpretation of urinary eosinophils. Arch Pathol Lab Med 113:1256–1258, 1989. 17. Schumann, GB: Utility of urinary cytology in renal diseases. Semin Nephrol 5(34) Sept, 1985. 18. Graber, M, et al: Bubble cells: Renal tubular cells in the urinary sediment with characteristics of viability. J Am Soc Nephrol 1(7):999–1004, 1991. 19. Baer, DM: Tips from clinical experts: Reporting of spermatozoa in microscopic urine exams. MLO 12:12, 1997. 20. Kumar, S, and Muchmore, A: Tamm-Horsfall protein— Uromodulin, 1950–1990. Kidney Int 37:1395–1399, 1990. 21. Haber, MH: Urinary Sediment: A Textbook Atlas. American Society of Clinical Pathologists, Chicago, 1981. 22. Lindner, LE, and Haber, MH: Hyaline casts in the urine: Mechanism of formation and morphological transformations. Am J Clin Pathol 80(3):347–352, 1983. 23. Lindner, LE, Jones, RN, and Haber, MH: A specific cast in acute pyelonephritis. Am J Clin Pathol 73(6):809–811, 1980. 24. Haber, MH, and Lindner, LE: The surface ultrastructure of urinary casts. Am J Clin Pathol 68(5):547–552, 1977. 25. Linder, LE, Vacca, D, and Haber, MF: Identification and composition of types of granular urinary cast. Am J Pathol 80(3):353–358, 1983. 3920_Ch06_099-146 23/01/14 10:19 AM Page 141 Chapter 6 | Microscopic Examination of Urine 141 Study Questions 1. Macroscopic screening of urine specimens is used to: A. Provide results as soon as possible B. Predict the type of urinary casts present C. Increase cost-effectiveness of urinalysis D. Decrease the need for polarized microscopy 2. Variations in the microscopic analysis of urine include all of the following except: A. Preparation of the urine sediment B. Amount of sediment analyzed C. Method of reporting D. Identification of formed elements 3. All of the following can cause false-negative microscopic results except: A. Braking the centrifuge B. Failing to mix the specimen C. Dilute alkaline urine D. Using midstream clean-catch specimens 4. The two factors that determine relative centrifugal force are: A. Radius of rotor head and rpm B. Radius of rotor head and time of centrifugation C. Diameter of rotor head and rpm D. RPM and time of centrifugation 5. When using the glass slide and cover-slip method, which of the following might be missed if the cover slip is overflowed? A. Casts B. RBCs C. WBCs D. Bacteria 6. Initial screening of the urine sediment is performed using an objective power of: A. 4× B. 10× C. 40× D. 100× 7. Which of the following should be used to reduce light intensity in bright-field microscopy? A. Centering screws B. Aperture diaphragm C. Rheostat D. Condenser aperture diaphragm 8. Which of the following are reported as number per lpf? A. RBCs B. WBCs C. Crystals D. Casts 9. The Sternheimer-Malbin stain is added to urine sediments to do all of the following except: A. Increase visibility of sediment constituents B. Change the constituents’ refractive index C. Decrease precipitation of crystals D. Delineate constituent structures 10. Nuclear detail can be enhanced by: A. Prussian blue B. Toluidine blue C. Acetic acid D. Both B and C 11. Which of the following lipids is/are stained by Sudan III? A. Cholesterol B. Neutral fats C. Triglycerides D. Both B and C 12. Which of the following lipids is/are capable of polarizing light? A. Cholesterol B. Neutral fats C. Triglycerides D. Both A and B 13. The purpose of the Hansel stain is to identify: A. Neutrophils B. Renal tubular cells C. Eosinophils D. Monocytes 14. Crenated RBCs are seen in urine that is: A. Hyposthenuric B. Hypersthenuric C. Highly acidic D. Highly alkaline 3920_Ch06_099-146 23/01/14 10:19 AM Page 142 142 Part Two | Urinalysis 15. Differentiation among RBCs, yeast, and oil droplets may be accomplished by all of the following except: 23. Forms of transitional epithelial cells include all of the following except: A. Observation of budding in yeast cells A. Spherical B. Increased refractility of oil droplets B. Caudate C. Lysis of yeast cells by acetic acid C. Convoluted D. Lysis of RBCs by acetic acid D. Polyhedral 16. A finding of dysmorphic RBCs is indicative of: A. Glomerular bleeding 24. Increased transitional cells are indicative of: A. Catheterization B. Renal calculi B. Malignancy C. Traumatic injury C. Pyelonephritis D. Coagulation disorders D. Both A and B 17. Leukocytes that stain pale blue with Sternheimer-Malbin stain and exhibit brownian movement are: 25. A primary characteristic used to identify renal tubular epithelial cells is: A. Indicative of pyelonephritis A. Elongated structure B. Basophils B. Centrally located nucleus C. Mononuclear leukocytes C. Spherical appearance D. Glitter cells D. Eccentrically located nucleus 18. Mononuclear leukocytes are sometimes mistaken for: A. Yeast cells B. Squamous epithelial cells C. Pollen grains D. Renal tubular cells 19. When pyuria is detected in a urine sediment, the slide should be carefully checked for the presence of: 26. Following an episode of hemoglobinuria, RTE cells may contain: A. Bilirubin B. Hemosiderin granules C. Porphobilinogen D. Myoglobin 27. The predecessor of the oval fat body is the: A. RBCs A. Histiocyte B. Bacteria B. Urothelial cell C. Hyaline casts C. Monocyte D. Mucus D. Renal tubular cell 20. Transitional epithelial cells are sloughed from the: A. Collecting duct B. Vagina C. Bladder D. Proximal convoluted tubule 21. The largest cells in the urine sediment are: A. Squamous epithelial cells B. Urothelial epithelial cells C. Cuboidal epithelial cells D. Columnar epithelial cells 22. A clinically significant squamous epithelial cell is the: A. Cuboidal cell B. Clue cell C. Caudate cell D. Columnar cell 28. A structure believed to be an oval fat body produced a Maltese cross formation under polarized light but does not stain with Sudan III. The structure: A. Contains cholesterol B. Is not an oval fat body C. Contains neutral fats D. Is contaminated with immersion oil 29. The finding of yeast cells in the urine is commonly associated with: A. Cystitis B. Diabetes mellitus C. Pyelonephritis D. Liver disorders 3920_Ch06_099-146 23/01/14 10:20 AM Page 143 Chapter 6 | Microscopic Examination of Urine 143 30. The primary component of urinary mucus is: 38. The presence of fatty casts is associated with: A. Bence Jones protein A. Nephrotic syndrome B. Microalbumin B. Crush injuries C. Uromodulin C. Diabetes mellitus D. Orthostatic protein D. All of the above 31. The majority of casts are formed in the: 39. Nonpathogenic granular casts contain: A. Proximal convoluted tubules A. Cellular lysosomes B. Ascending loop of Henle B. Degenerated cells C. Distal convoluted tubules C. Protein aggregates D. Collecting ducts D. Gram-positive cocci 32. Cylindruria refers to the presence of: 40. All of the following are true about waxy casts except they: A. Cylindrical renal tubular cells A. Represent extreme urine stasis B. Mucus-resembling casts B. May have a brittle consistency C. Hyaline and waxy casts C. Require staining to be visualized D. All types of casts D. Contain degenerated granules 33. A person submitting a urine specimen following a strenuous exercise routine can normally have all of the following in the sediment except: 41. Observation of broad casts represents: A. Destruction of tubular walls A. Hyaline casts B. Dehydration and high fever B. Granular casts C. Formation in the collecting ducts C. RBC casts D. Both A and C D. WBC casts 34. Prior to identifying an RBC cast, all of the following should be observed except: 42. All of the following contribute to urinary crystals formation except: A. Protein concentration A. Free-floating RBCs B. pH B. Intact RBCs in the cast C. Solute concentration C. Presence of a cast matrix D. Temperature D. A positive reagent strip blood reaction 35. WBC casts are primarily associated with: 43. The most valuable initial aid for identifying crystals in a urine specimen is: A. Pyelonephritis A. pH B. Cystitis B. Solubility C. Glomerulonephritis C. Staining D. Viral infections D. Polarized microscopy 36. The shape of the RTE cell associated with renal tubular epithelial casts is primarily: 44. Crystals associated with severe liver disease include all of the following except: A. Elongated A. Bilirubin B. Cuboidal B. Leucine C. Round C. Cystine D. Columnar D. Tyrosine 37. When observing RTE casts, the cells are primarily: 45. All of the following crystals routinely polarize except: A. Embedded in a clear matrix A. Uric acid B. Embedded in a granular matrix B. Cholesterol C. Attached to the surface of a matrix C. Radiographic dye D. Stained by components of the urine filtrate D. Cystine 3920_Ch06_099-146 23/01/14 10:20 AM Page 144 144 Part Two | Urinalysis 46. Casts and fibers can usually be differentiated using: A. Solubility characteristics B. Patient history C. Polarized light D. Fluorescent light 47. Match the following crystals seen in acidic urine with their description/identifying characteristics: ____ Amorphous urates 1. Envelopes ____ Uric acid 2. Thin needles ____ Calcium oxalate monohydrate 3. Yellow-brown, whetstone ____ Calcium oxalate dihydrate 4. Pink sediment 5. Ovoid 48. Match the following crystals seen in alkaline urine with their description/identifying characteristics: ____ Triple phosphate 1. Yellow granules ____ Amorphous phosphate 2. Thin prisms ____ Calcium phosphate 3. “Coffin lids” ____ Ammonium biurate 4. Dumbbell shape ____ Calcium carbonate 5. White precipitate 6. Thorny apple 49. Match the following abnormal crystals with their description/identifying characteristics: ____ Cystine 1. Bundles following refrigeration ____ Tyrosine 2. Highly alkaline pH ____ Cholesterol 3. Bright yellow clumps ____ Leucine 4. Hexagonal plates ____ Ampicillin 5. Flat plates, high specific gravity ____ Radiographic dye 6. Concentric circles, radial striations ____ Bilirubin 7. Notched corners 8. Fine needles seen in liver disease 50. Match the following types of microscopy with their descriptions: ____ Bright-field 1. Indirect light is reflected off the object ____ Phase 2. Objects split light into two beams ____ Polarized 3. Low refractive index objects may be overlooked ____ Dark-field 4. Three-dimensional images ____Fluorescent 5. Forms halo of light around object ____Interference 6. Detects electrons contrast emitted from objects 7. Detects specific wavelengths of light emitted from objects 3920_Ch06_099-146 23/01/14 10:20 AM Page 145 Chapter 6 | Microscopic Examination of Urine 145 Case Studies and Clinical Situations 1. An 85-year-old woman with diabetes and a broken hip has been confined to bed for the past 3 months. Results of an ancillary blood glucose test are 250 mg/dL, and her physician orders additional blood tests and a routine urinalysis. The urinalysis report is as follows: COLOR: Pale yellow KETONES: Negative CLARITY: Hazy BLOOD: Moderate SP. GRAVITY: 1.020 BILIRUBIN: Negative pH: 5.5 UROBILINOGEN: Normal PROTEIN: Trace NITRITE: Negative GLUCOSE: 100 mg/dL LEUKOCYTES: 2+ Microscopic: 20 to 25 WBCs/hpf Many yeast cells and hyphae a. Why are yeast infections common in patients with diabetes mellitus? b. With a blood glucose level of 250 mg/dL, should glucose be present in the urine? Why or why not? c. Is there a discrepancy between the negative nitrite and the positive leukocyte esterase results? Explain your answer. d. What is the major discrepancy between the chemical and microscopic results? e. Considering the patient’s history, what is the most probable cause for the discrepancy? 2. A medical technology student training in a newly renovated STAT laboratory is having difficulty performing a microscopic urinalysis. Reagent strip testing indicates the presence of moderate blood and leukocytes, but the student is also observing some large unusual objects resembling crystals and possible casts. The student is also having difficulty keeping all of the constituents in focus at the same time. a. Why is the student having difficulty focusing? b. What is a possible cause of the unusual microscopic constituents? c. Should the student be concerned about the unusual microscopic constituents? Explain your answer. d. What microscopy technique could be used to aid in differentiating a cast and an artifact? 3. A prisoner sentenced to 10 years for selling illegal drugs develops jaundice, lethargy, and hepatomegaly. A test for hepatitis B surface antigen is positive, and the patient is placed in the prison infirmary. When his condition appears to worsen and a low urinary output is observed, the patient is transferred to a local hospital. Additional testing detects a superinfection with delta hepatitis virus and decreased renal concentrating ability. Urinalysis results are as follows: COLOR: Amber KETONES: Negative CLARITY: Hazy BLOOD: Negative SP. GRAVITY: 1.011 BILIRUBIN: Large pH: 7.0 UROBILINOGEN: 4.0 EU PROTEIN: 2+ NITRITE: Negative GLUCOSE: Negative LEUKOCYTES: Negative Microscopic: 2 to 4 WBCs/hpf 1 to 2 hyaline casts/lpf 1 to 3 RBCs/hpf 1 to 2 granular casts/lpf 2 to 4 bile-stained RTE cells/hpf 0 to 1 RTE casts/lpf 0 to 1 bile-stained waxy casts/lpf a. Based on the urinalysis results, in what area of the nephron is damage occurring? b. Is this consistent with the patient’s primary diagnosis? Explain your answer. c. What is causing the RTE cells to be bile stained? d. Why is the urobilinogen level elevated? e. State a disorder in which the urobilinogen level would be elevated, but the bilirubin result would be negative. 4. A 30-year-old woman being treated for a UTI brings a urine specimen to the Employee Health Clinic at 4:00 p.m. The nurse on duty tells her that the specimen will be refrigerated and tested by the technologist the next morning. The technologist has difficulty interpreting the color of the reagent strip tests and reports only the following results: COLOR: Amber CLARITY: Slightly cloudy Microscopic: 3 to 5 RBCs/hpf 8 to 10 WBCs/hpf Moderate bacteria Moderate colorless crystals appearing in bundles a. What could have caused the technologist to have difficulty interpreting the reagent strip results? b. Could this specimen produce a yellow foam when shaken? c. What could the technologist do to aid in the identification of the crystals? d. What is the probable identification of the colorless crystals? 3920_Ch06_099-146 23/01/14 10:20 AM Page 146 146 Part Two | Urinalysis 5. A 2-year-old left unattended in the garage for 5 minutes is suspected of ingesting antifreeze (ethylene glycol). The urinalysis has a pH of 6.0 and is negative on the chemical examination. Two distinct forms of crystals are observed in the microscopic examination. a. What type of crystals would you expect to be present? b. What is the other form of this crystal? c. Describe the two forms. d. Which form would you expect to be predominant? 6. A female patient comes to the outpatient clinic with symptoms of UTI. She brings a urine specimen with her. Results of the routine analysis performed on this specimen are as follows: COLOR: Yellow KETONES: Negative CLARITY: Hazy BLOOD: Small SP. GRAVITY: 1.015 BILIRUBIN: Negative pH: 9.0 UROBILINOGEN: Normal PROTEIN: Negative NITRITE: Negative GLUCOSE: Negative LEUKOCYTE: 2+ Microscopic: 1 to 3 RBCs/hpf Heavy bacteria 8 to 10 WBCs/hpf Moderate squamous epithelial cells a. What discrepancies are present between the chemical and microscopic test results? b. State a reason for the discrepancies. c. Identify a chemical result in the urinalysis that confirms your reason for the discrepancies. d. What course of action should the laboratory take to obtain accurate results for this patient? 7. A high school student is taken to the emergency room with a broken leg that occurred during a football game. The urinalysis results are as follows: COLOR: Dark yellow KETONES: Negative CLARITY: Hazy BLOOD: Moderate SP. GRAVITY: 1.030 BILIRUBIN: Negative pH: 5.5 UROBILINOGEN: Normal PROTEIN: 2+ NITRITE: Negative GLUCOSE: Negative LEUKOCYTE: Negative Microscopic: 0 to 3 WBCs/hpf 0 to 4 hyaline casts/lpf 0 to 3 granular casts/lpf Few squamous epithelial cells a. Are these results of clinical significance? b. Explain the discrepancy between the chemical and microscopic blood results. c. What is the probable cause of the granular casts? 8. As supervisor of the urinalysis section, you are reviewing results. State why or why not each of the following results would concern you. a. The presence of waxy casts and a negative protein in urine from a 6–month-old girl b. Increased transitional epithelial cells in a specimen obtained following cystoscopy c. Tyrosine crystals in a specimen with a negative bilirubin test result d. Cystine crystals in a specimen from a patient diagnosed with gout e. Cholesterol crystals in urine with a specific gravity greater than 1.040 f. Trichomonas vaginalis in a male urine specimen g. Amorphous urates and calcium carbonate crystals in a specimen with a pH of 7.0 3920_Ch07_147-162 23/01/14 10:21 AM Page 147 CHAPTER 7 Renal Disease LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 7-1 Differentiate among renal diseases of glomerular, tubular, interstitial, and vascular origin. 7-2 Describe the processes by which immunologic damage is caused to the glomerular basement membrane. 7-3 Define glomerulonephritis. 7-4 Describe the characteristic clinical symptoms, etiology, and urinalysis findings in acute poststreptococcal and rapidly progressive glomerulonephritis, Goodpasture syndrome, Wegener granulomatosis, and Henoch-Schönlein purpura. 7-5 Name three renal disorders that also involve acute respiratory symptoms. 7-9 Compare and contrast the nephrotic syndrome and minimal change disease with regard to laboratory results and course of disease. 7-10 State two causes of acute tubular necrosis. 7-11 Name the urinary sediment constituent most diagnostic of renal tubular damage. 7-12 Describe Fanconi syndrome, Alport syndrome, uromodulin-associated renal disease, and renal glucosuria. 7-13 Differentiate between diabetic nephropathy and nephrogenic diabetes insipidus. 7-6 Differentiate between membranous and membranoproliferative glomerulonephritis. 7-14 Compare and contrast the urinalysis results in patients with cystitis, pyelonephritis, and acute interstitial nephritis. 7-7 Discuss the clinical course and significant laboratory results associated with immunoglobulin A nephropathy. 7-15 Differentiate among causes of laboratory results associated with prerenal, renal, and postrenal acute renal failure. 7-8 Relate laboratory results associated with the nephrotic syndrome to the disease process. 7-16 Discuss the formation of renal calculi, composition of renal calculi, and patient management techniques. KEY TERMS Acute interstitial nephritis (AIN) Focal segmental glomerulosclerosis (FSGS) Pyelonephritis Antiglomerular basement membrane antibody (ANCA) Glomerulonephritis Uromodulin-associated kidney disease Antineutrophilic cytoplasmic antibody Lithiasis Cystitis Nephrotic syndrome Acute tubular necrosis (ATN) IgA nephropathy Lithotripsy Tubulointerstitial disease 3920_Ch07_147-162 23/01/14 10:21 AM Page 148 148 Part Two | Urinalysis Disorders throughout the body can affect renal function and produce abnormalities in the urinalysis. Considering that the major function of the kidneys is filtration of the blood to remove waste products, it becomes evident that the kidneys are consistently exposed to potentially damaging substances. Renal disease is often classified as being glomerular, tubular, or interstitial, based on the area of the kidney primarily affected. In this chapter, we will cover the most commonly encountered disorders in relation to the affected areas of the kidney. Basic knowledge of these disorders can be helpful when analyzing the results of a routine urinalysis. Glomerular Disorders Most glomerular disorders result from immunologic disorders throughout the body, including the kidney. Immune complexes formed as a result of immunologic reactions and increased serum immunoglobulins, such as immunoglobulin A (IgA), circulate in the bloodstream and are deposited on the glomerular membranes. Components of the immune system, including complement, neutrophils, lymphocytes, monocytes, and cytokines, are then attracted to the area, producing changes and damage to the membranes. Depending on the immune system mediators involved, damage may consist of cellular infiltration or proliferation resulting in thickening of the glomerular basement membrane, and complement-mediated damage to the capillaries and basement membrane. Nonimmunologic causes of glomerular damage include exposure to chemicals and toxins that also affect the tubules, disruption of the electrical membrane charges as occurs in the nephrotic syndrome, deposition of amyloid material from systemic disorders that may involve chronic inflammation and acute-phase reactants, and the basement membrane thickening associated with diabetic nephropathy. Glomerulonephritis The term glomerulonephritis refers to a sterile, inflammatory process that affects the glomerulus and is associated with the finding of blood, protein, and casts in the urine.1 A variety of types of glomerulonephritis exist, and the condition also may progress from one form to another (i.e., rapidly progressive glomerular nephritis to chronic glomerulonephritis to the nephrotic syndrome and eventual renal failure). Acute Poststreptococcal Glomerulonephritis As its name implies, acute glomerulonephritis (AGN) is a disease marked by the sudden onset of symptoms consistent with damage to the glomerular membrane. These may include fever, edema (most noticeably around the eyes), fatigue, hypertension, oliguria, and hematuria. Symptoms usually occur in children and young adults following respiratory infections caused by certain strains of group A streptococcus that contain M protein in the cell wall. During the course of the infection, these nephrogenic strains of streptococci form immune complexes with their corresponding circulating antibodies and become deposited on the glomerular membranes. The accompanying inflammatory reaction affects glomerular function. In most cases, successful management of the secondary complications (hypertension and electrolyte imbalance) until the immune complexes have been cleared from the blood and the inflammation subsides, results in no permanent kidney damage. Primary urinalysis findings include marked hematuria, proteinuria, and oliguria, accompanied by red blood cell (RBC) casts, dysmorphic RBCs, hyaline and granular casts, and white blood cells (WBCs). As toxicity to the glomerular membrane subsides, urinalysis results return to normal, with the possible exception of microscopic hematuria that lasts until the membrane damage has been repaired. Blood urea nitrogen (BUN) may be elevated during the acute stages but, like the urinalysis, returns to normal. Demonstration of positive anti–group A streptococcal enzyme tests provides evidence that the disease is of streptococcal origin. Since the development of rapid anti–group A streptococcal enzyme tests that can be performed in a physician’s office, urgent care facility, or emergency department, the incidence of acute post-streptococcal glomerulonephritis has declined. Rapidly Progressive (Crescentic) Glomerulonephritis A more serious form of acute glomerular disease is called rapidly progressive (or crescentic) glomerulonephritis and has a much poorer prognosis, often terminating in renal failure. Symptoms are initiated by deposition of immune complexes in the glomerulus, often as a complication of another form of glomerulonephritis or an immune systemic disorder such as systemic lupus erythematosus (SLE). Damage by macrophages to the capillary walls releases cells and plasma into Bowman’s space, and the production of crescentic formations containing macrophages, fibroblasts, and polymerized fibrin, causes permanent damage to the capillary tufts. Initial laboratory results are similar to acute glomerulonephritis but become more abnormal as the disease progresses, including markedly elevated protein levels and very low glomerular filtration rates. Some forms may demonstrate increased fibrin degradation products, cryoglobulins, and the deposition of IgA immune complexes in the glomerulus.2 Goodpasture Syndrome Morphologic changes to the glomeruli resembling those in rapidly progressive glomerular nephritis are seen in conjunction with the autoimmune disorder termed Goodpasture syndrome. A cytotoxic autoantibody can appear against the glomerular and alveolar basement membranes after viral respiratory infections. Attachment of this autoantibody to the basement membrane, followed by complement activation, produces the capillary destruction. Referred to as antiglomerular basement membrane antibody, the autoantibody can be detected in patient serum. Initial pulmonary complaints are hemoptysis and dyspnea, followed by the development of hematuria. Urinalysis 3920_Ch07_147-162 23/01/14 10:21 AM Page 149 Chapter 7 | Renal Disease results include proteinuria, hematuria, and the presence of RBC casts. Progression to chronic glomerulonephritis and endstage renal failure is common. Wegener Granulomatosis Wegener granulomatosis causes a granuloma-producing inflammation of the small blood vessels primarily of the kidney and respiratory system. Key to the diagnosis of Wegener granulomatosis is the demonstration of antineutrophilic cytoplasmic antibody (ANCA) in the patient’s serum.3 Binding of these autoantibodies to the neutrophils located in the vascular walls may initiate the immune response and the resulting granuloma formation. Patients usually present first with pulmonary symptoms and later develop renal involvement, including hematuria, proteinuria, RBC casts, and elevated serum creatinine and BUN. Testing for ANCA includes incubating the patient’s serum with either ethanol or formalin/formaldehyde-fixed neutrophils and examining the preparation using indirect immunofixation to detect the serum antibodies attached to the neutrophils. If the neutrophils are fixed in ethanol, the antibodies form a perinuclear pattern called p-ANCA. When the neutrophils are fixed with formalin/formaldehyde, the pattern is granular throughout the cytoplasm called c-ANCA.4 Henoch-Schönlein Purpura Henoch-Schönlein purpura disease occurs primarily in children after upper respiratory infections. As its name implies, initial symptoms include the appearance of raised, red patches on the skin. Respiratory and gastrointestinal symptoms, including blood in the sputum and stools, may be present. Renal involvement is the most serious complication of the disorder and may range from mild to heavy proteinuria and hematuria with RBC casts. Complete recovery with normal renal function is seen in more than 50% of patients. In other patients, progression to a more serious form of glomerulonephritis and renal failure may occur. Urinalysis and renal function assessment should be used to monitor patients following recovery from the original symptoms. Membranous Glomerulonephritis The predominant characteristic of membranous glomerulonephritis is a pronounced thickening of the glomerular basement membrane resulting from the deposition of immunoglobulin G immune complexes. Disorders associated with membranous glomerulonephritis development include systemic lupus erythematosus, Sjögren syndrome, secondary syphilis, hepatitis B, gold and mercury treatments, and malignancy. Many cases of unknown etiology have been reported. As a rule, the disease progresses slowly, with possible remission; however, nephrotic syndrome symptoms frequently develop.5 There may also be a tendency toward thrombosis. Laboratory findings include microscopic hematuria and elevated urine protein excretion that may reach concentrations similar to those in the nephrotic syndrome. Demonstration of 149 one of the secondary disorders through blood tests can aid in the diagnosis. Membranoproliferative Glomerulonephritis Membranoproliferative glomerulonephritis (MPGN) is marked by two different alterations in the cellularity of the glomerulus and peripheral capillaries. Type 1 displays increased cellularity in the subendothelial cells of the mesangium (interstitial area of Bowman’s capsule), causing thickening of the capillary walls, whereas type 2 displays extremely dense deposits in the glomerular basement membrane. Many of the patients are children, and the disease has a poor prognosis: type 1 patients progress to the nephrotic syndrome and type 2 patients experience symptoms of chronic glomerulonephritis. The laboratory findings vary, but hematuria, proteinuria, and decreased serum complement levels are usual findings. There appears to be an association with autoimmune disorders, infections, and malignancies.6 Chronic Glomerulonephritis Depending on the amount and duration of the damage to the glomerulus in the previously discussed glomerular disorders, progression to chronic glomerulonephritis and end-stage renal disease may occur. Gradually worsening symptoms include fatigue, anemia, hypertension, edema, and oliguria. Examination of the urine reveals hematuria, proteinuria, glucosuria as a result of tubular dysfunction, and many varieties of casts, including broad casts. A markedly decreased glomerular filtration rate is present in conjunction with increased BUN and creatinine levels and electrolyte imbalance. Immunoglobulin A Nephropathy Also known as Berger disease, IgA nephropathy, in which immune complexes containing IgA are deposited on the glomerular membrane, is the most common cause of glomerulonephritis. Patients have increased serum levels of IgA, which may be a result of a mucosal infection. The disorder is most frequently seen in children and young adults. Patients usually present with an episode of macroscopic hematuria following an infection or strenuous exercise. Recovery from the macroscopic hematuria is spontaneous; however, asymptomatic microhematuria and elevated serum levels of IgA remain.7 Except for periodic episodes of macroscopic hematuria, a patient with the disorder may remain essentially asymptomatic for 20 years or more; however, there is a gradual progression to chronic glomerulonephritis and end-stage renal disease. Nephrotic Syndrome The nephrotic syndrome is marked by massive proteinuria (greater than 3.5 g/day), low levels of serum albumin, high levels of serum lipids, and pronounced edema.1 Acute onset of the disorder can occur in instances of circulatory disruption producing systemic shock that decrease the pressure and flow of blood to the kidney. Progression to the nephrotic syndrome may also 3920_Ch07_147-162 23/01/14 10:21 AM Page 150 150 Part Two | Urinalysis occur as a complication of the previously discussed forms of glomerulonephritis. Increased permeability of the glomerular membrane is attributed to damage to the shield of negativity and the podocytes that produces a less tightly connected barrier. Such damage facilitates passage of high-molecular-weight proteins and lipids and negatively charged albumin into the urine. Albumin is the primary protein depleted from the circulation. The ensuing hypoalbuminemia appears to stimulate increased lipid production by the liver. The lower oncotic pressure in the capillaries resulting from the depletion of plasma albumin increases fluid loss into the interstitial spaces, which, accompanied by sodium retention, produces the edema. Depletion of immunoglobulins and coagulation factors places patients at an increased risk of infection and coagulation disorders. Both tubular and glomerular damage occurs, and the nephrotic syndrome may progress to chronic renal failure. Urinalysis observations include marked proteinuria; urinary fat droplets; oval fat bodies; renal tubular epithelial (RTE) cells; epithelial, fatty, and waxy casts; and microscopic hematuria. Absorption of the lipid-containing proteins by the RTE cells followed by cellular sloughing produces the characteristic oval fat bodies seen in the sediment examination. Minimal Change Disease As the name implies, minimal change disease (also known as lipid nephrosis) produces little cellular change in the glomerulus, except for some damage to the podocytes and the shield of negativity, allowing for increased protein filtration. Patients are usually children who present with edema, heavy proteinuria, transient hematuria, and normal BUN and creatinine results. Although the etiology is unknown at this time, allergic reactions, recent immunization, and possession of the human leukocyte antigen-B12 (HLA-B12) antigen have been associated with this disease. The disorder responds well to corticosteroids, and prognosis is generally good, with frequent complete remissions.8 Focal Segmental Glomerulosclerosis In contrast to the previously discussed disorders, focal segmental glomerulosclerosis (FSGS) affects only certain Table 7–1 numbers and areas of glomeruli, and the others remain normal. Symptoms may be similar to the nephrotic syndrome and minimal change disease owing to damaged podocytes. Immune deposits, primarily immunoglobulin M and C3, are a frequent finding and can be seen in undamaged glomeruli. FSGS is often seen in association with abuse of heroin and analgesics and with AIDS. Moderate to heavy proteinuria and microscopic hematuria are the most consistent urinalysis findings. Laboratory testing and clinical information for the glomerular disorders are summarized in Tables 7–1 and 7–2. Tubular Disorders Disorders affecting the renal tubules include those in which tubular function is disrupted as a result of actual damage to the tubules and those in which a metabolic or hereditary disorder affects the intricate functions of the tubules. Acute Tubular Necrosis The primary disorder associated with damage to the renal tubules is acute tubular necrosis (ATN). Damage to the RTE cells may be produced by decreased blood flow that causes a lack of oxygen presentation to the tubules (ischemia) or the presence of toxic substances in the urinary filtrate. Disorders causing ischemic ATN include shock, trauma (such as crushing injuries), and surgical procedures. “Shock” is a general term indicating a severe condition that decreases the flow of blood throughout the body. Examples of conditions that may cause shock are cardiac failures, sepsis involving toxigenic bacteria, anaphylaxis, massive hemorrhage, and contact with high-voltage electricity. Exposure to a variety of nephrotoxic agents can damage and affect the function of the RTE cells. Substances include aminoglycoside antibiotics, the antifungal agent amphotericin B, cyclosporine, radiographic dye, organic solvents such as ethylene glycol, heavy metals, and toxic mushrooms. As discussed in Chapter 5, filtration of large amounts of hemoglobin and myoglobin is also nephrotoxic. Laboratory Testing in Glomerular Disorders Disorder Primary Urinalysis Result Other Significant Tests Acute glomerulonephritis Macroscopic hematuria Anti–group A streptococcal enzyme tests Rapidly progressive glomerulonephritis Proteinuria RBC casts Granular casts Macroscopic hematuria Proteinuria RBC casts BUN Creatinine eGFR 3920_Ch07_147-162 23/01/14 10:21 AM Page 151 Chapter 7 | Renal Disease Table 7–1 Laboratory Testing in Glomerular Disorders—cont’d Disorder Primary Urinalysis Result Other Significant Tests Goodpasture syndrome Macroscopic hematuria Antiglomerular basement membrane antibody Wegener granulomatosis Proteinuria RBC casts Macroscopic hematuria Henoch-Schönlein purpura Membranous glomerulonephritis Membranoproliferative glomerulonephritis Chronic glomerulonephritis IgA nephropathy (early stages) IgA nephropathy (late stages) Nephrotic syndrome Minimal change disease Focal segmental glomerulosclerosis Alport syndrome (early stages) (late stages) Diabetic nephropathy (late stages) Proteinuria RBC casts Macroscopic hematuria Proteinuria RBC casts Microscopic hematuria Proteinuria Hematuria Proteinuria Hematuria Proteinuria Glucosuria Cellular and granular casts Waxy and broad casts Macroscopic or microscopic hematuria See Chronic glomerulonephritis Heavy proteinuria Microscopic hematuria Renal tubular cells Oval fat bodies Fat droplets Fatty and waxy casts Heavy proteinuria Transient hematuria Fat droplets Proteinuria Microscopic hematuria Macroscopic or microscopic hematuria See Nephrotic syndrome Microalbuminuria See Chronic glomerulonephritis Antineutrophilic peripheral or cytoplasmic antibody Stool occult blood Antinuclear antibody Hepatitis B surface antigen Fluorescent treponemal antibody-absorption test (FTA-ABS) Serum complement levels BUN Serum creatinine eGFR Electrolytes Serum IgA Serum albumin Cholesterol Triglycerides Serum albumin Cholesterol Triglycerides Drugs of abuse HIV tests Genetic testing Blood glucose 151 3920_Ch07_147-162 23/01/14 10:21 AM Page 152 152 Part Two | Urinalysis Table 7–2 Clinical Information Associated With Glomerular Disorders Disorder Etiology Clinical Course Acute glomerulonephritis Deposition of immune complexes, formed in conjunction with group A Streptococcus infection, on the glomerular membranes Deposition of immune complexes from systemic immune disorders on the glomerular membrane Attachment of a cytotoxic antibody formed during viral respiratory infections to glomerular and alveolar basement membranes Rapid onset of hematuria and edema, permanent renal damage seldom occurs Rapid onset with glomerular damage and possible progression to end-stage renal failure Hemoptysis and dyspnea followed by hematuria Rapidly progressive glomerulonephritis Goodpasture syndrome Wegener granulomatosis Antineutrophilic cytoplasmic autoantibody binds to neutrophils in vascular walls producing damage to small vessels in the lungs and glomerulus Henoch-Schönlein purpura Occurs primarily in children following viral respiratory infections; a decrease in platelets disrupts vascular integrity Membranous glomerulonephritis Thickening of the glomerular membrane following IgG immune complex deposition associated with systemic disorders Cellular proliferation affecting the capillary walls or the glomerular basement membrane, possibly immune-mediated Marked decrease in renal function resulting from glomerular damage precipitated by other renal disorders Deposition of IgA on the glomerular membrane resulting from increased levels of serum IgA Membranoproliferative glomerulonephritis Chronic glomerulonephritis IgA nephropathy Nephrotic syndrome Disruption of the shield of negativity and damage to the tightly fitting podocyte barrier resulting in massive loss of protein and lipids Minimal change disease Disruption of the podocytes occurring primarily in children following allergic reactions and immunizations Disruption of podocytes in certain areas of glomeruli associated with heroin and analgesic abuse and AIDS Genetic disorder showing lamellated and thinning glomerular basement membrane Focal segmental glomerulosclerosis Alport syndrome Possible progression to end-stage renal failure Pulmonary symptoms including hemoptysis develop first followed by renal involvement and possible progression to end-stage renal failure Initial appearance of purpura followed by blood in sputum and stools and eventual renal involvement Complete recovery is common, but may progress to renal failure Slow progression to the nephrotic syndrome or possible remission Slow progression to chronic glomerulonephritis or nephrotic syndrome Noticeable decrease in renal function progressing to renal failure Recurrent macroscopic hematuria following exercise with slow progression to chronic glomerulonephritis Acute onset following systemic shock Gradual progression from other glomerular disorders and then to renal failure Frequent complete remission following corticosteroid treatment May resemble nephrotic syndrome or minimal change disease Slow progression to nephrotic syndrome and end-stage renal disease 3920_Ch07_147-162 23/01/14 10:21 AM Page 153 Chapter 7 | Renal Disease The disease course of ATN varies. It may present as an acute complication of an ischemic event or more gradually during exposure to toxic agents. Correcting the ischemia, removing the toxic substances, and effectively managing the accompanying symptoms of acute renal failure frequently result in a complete recovery. Urinalysis findings include mild proteinuria, microscopic hematuria, and, most noticeably, the presence of RTE cells and RTE cell casts containing tubular fragments consisting of three or more cells. As a result of the tubular damage, a variety of other casts may be present, including hyaline, granular, waxy, and broad. Hereditary and Metabolic Tubular Disorders Disorders affecting tubular function may be caused by systemic conditions that affect or override the tubular reabsorptive maximum (Tm) for particular substances normally reabsorbed by the tubules or by failure to inherit a gene or genes required for tubular reabsorption. Fanconi Syndrome The disorder most frequently associated with tubular dysfunction is Fanconi syndrome. The syndrome consists of a generalized failure of tubular reabsorption in the proximal convoluted tubule. Therefore, substances most noticeably affected include glucose, amino acids, phosphorous, sodium, potassium, bicarbonate, and water. Tubular reabsorption may be affected by dysfunction of the transport of filtered substances across the tubular membranes, disruption of cellular energy needed for transport, or changes in the tubular membrane permeability. Fanconi syndrome may be inherited in association with cystinosis and Hartnup disease (see Chapter 8), acquired through exposure to toxic agents, including heavy metals and outdated tetracycline, or seen as a complication of multiple myeloma and renal transplant. Urinalysis findings include glycosuria with a normal blood glucose and possible mild proteinuria. Urinary pH can be very low due to the failure to reabsorb bicarbonate. Alport Syndrome Alport syndrome is an inherited disorder of collagen production affecting the glomerular basement membrane. The syndrome can be inherited as a sex-linked or autosomal genetic disorder. Males inheriting the X-linked gene are more severely affected than females inheriting the autosomal gene. During respiratory infections, males younger than age 6 years may exhibit macroscopic hematuria and continue to exhibit microscopic hematuria. Abnormalities in hearing and vision may also develop. The glomerular basement membrane has a lamellated appearance with areas of thinning. No evidence of glomerular antibodies is present. The prognosis ranges from mild symptoms to persistent hematuria and renal insufficiency in later life to the nephrotic syndrome and end-stage renal disease. 153 Uromodulin-Associated Kidney Disease Uromodulin is a glycoprotein and is the only protein produced by the kidney. It is produced by the proximal and distal convoluted tubules. Although it is not measured by routine laboratory methods, research has shown that it is the primary protein found in normal urine. Uromodulin-associated kidney disease is primarily an inherited disorder caused by an autosomal mutation in the gene that produces uromodulin. The mutation causes a decrease in the production of normal uromodulin that is replaced by the abnormal form. The abnormal uromodulin is still produced by the tubular cells and accumulates in these cells, resulting in their destruction, which leads to the need for renal monitoring and eventual renal transplantation. 9 The mutation also causes an increase in serum uric acid, resulting in persons developing gout as early as the teenage years before the onset of detectable renal disease.9 Diabetic Nephropathy Diabetic nephropathy is currently the most common cause of end-stage renal disease. Damage to the glomerular membrane occurs not only as a result of glomerular membrane thickening but also because of the increased proliferation of mesangial cells and increased deposition of cellular and noncellular material within the glomerular matrix, resulting in accumulation of solid substances around the capillary tufts. This glomerular damage is believed to be associated with deposition of glycosylated proteins resulting from poorly controlled blood glucose levels. The vascular structure of the glomerulus also develops sclerosis. As discussed in Chapter 5, early monitoring of persons diagnosed with diabetes mellitus for the presence of microalbuminuria is important to detect the onset of diabetic nephropathy. Modification of diet and strict control of hypertension can decrease the progression of the renal disease. Nephrogenic Diabetes Insipidus As discussed in Chapter 3, urine concentration is regulated in the distal convoluted tubules and the collecting ducts in response to antidiuretic hormone (ADH) produced by the hypothalamus. When the action of ADH is disrupted either by the inability of the renal tubules to respond to ADH (nephrogenic diabetes insipidus [DI]) or failure of the hypothalamus to produce ADH (neurogenic DI), excessive amounts of urine are excreted. Differentiation between the two types of DI is covered in Chapter 3. Nephrogenic DI can be inherited as a sex-linked recessive gene or acquired from medications, including lithium and amphotericin B. It also may be seen as a complication of polycystic kidney disease and sickle cell anemia. TECHNICAL TIP As discussed in Chapter 6, uromodulin forms the matrix of urinary casts seen in many renal disorders. The defective gene is not associated with other renal disorders. 3920_Ch07_147-162 23/01/14 10:21 AM Page 154 154 Part Two | Urinalysis Urinalysis findings associated with DI are low specific gravity, pale yellow color, and possible false-negative results for chemical tests. Renal Glycosuria In contrast to Fanconi syndrome, which exhibits a generalized failure to reabsorb substances from the glomerular filtrate, renal glucosuria affects only the reabsorption of glucose. The disorder is inherited as an autosomal recessive trait. In inherited renal glucosuria either the number of glucose transporters in the tubules is decreased or the affinity of the transporters for glucose is decreased. Under normal conditions glucose is not present in the urine unless the blood glucose level reaches the maximal tubular reabsorption capacity for glucose (TMG), which is 160 to 180 mg/dL. Patients with renal glycosuria have increased urine glucose concentrations with normal blood glucose concentrations. Table 7–3 Laboratory testing and clinical information for the hereditary and metabolic disorders are summarized in Tables 7–3 and 7–4. Interstitial Disorders Considering the close proximity between the renal tubules and the renal interstitium, disorders affecting the interstitium also affect the tubules, resulting in the condition commonly called tubulointerstitial disease. Most of these disorders involve infections and inflammatory conditions. The most common renal disease is a urinary tract infection (UTI). Infection may involve the lower urinary tract (urethra and bladder) or the upper urinary tract (renal pelvis, tubules, and interstitium). Most frequently encountered is infection of the bladder (cystitis), which can Laboratory Testing in Metabolic and Hereditary Tubular Disorders Disorder Primary Urinalysis Results Other Significant Tests Acute tubular necrosis Microscopic hematuria Proteinuria Renal tubular epithelial cells Renal tubular epithelial cell casts Hyaline, granular, waxy, broad casts Glucosuria Possible cystine crystals Renal tubular epithelial cells Hemoglobin Hematocrit Fanconi syndrome Uromodulin-associated kidney disease (early stages) Late stages Nephrogenic diabetes insipidus Renal glucosuria Table 7–4 Cardiac enzymes Serum and urine electrolytes Amino acid chromatography Serum uric acid See chronic glomerulonephritis Low specific gravity, polyuria Glucosuria ADH testing Blood glucose Clinical Information Associated With Metabolic and Tubular Disorders Disorder Etiology Clinical Course Acute tubular necrosis Damage to renal tubular cells caused by ischemia or toxic agents Fanconi syndrome Inherited in association with cystinosis and Hartnup disease or acquired through exposure to toxic agents Inherited defect in the production of normal uromodulin by the renal tubules and increased uric acid causing gout Inherited defect of tubular response to ADH or acquired from medications Inherited autosomal recessive trait Acute onset of renal dysfunction usually resolved when underlying cause is corrected Generalized defect in renal tubular reabsorption requiring supportive therapy Uromodulin-associated kidney disease Nephrogenic diabetes insipidus Renal glucosuria Continual monitoring of renal function for progression to renal failure and possible kidney transplantation Requires supportive therapy to prevent dehydration Benign disorder 3920_Ch07_147-162 23/01/14 10:21 AM Page 155 Chapter 7 | Renal Disease progress to a more serious upper UTI if left untreated. Cystitis is seen more often in women and children, who present with symptoms of urinary frequency and burning. Urinalysis reveals the presence of numerous WBCs and bacteria, often accompanied by mild proteinuria and hematuria and an increased pH. Acute Pyelonephritis Infection of the upper urinary tract, including both the tubules and interstitium, is termed pyelonephritis and can occur in both acute and chronic forms. Acute pyelonephritis most frequently occurs as a result of ascending movement of bacteria from a lower UTI into the renal tubules and interstitium. Patients present with rapid onset of symptoms, including urinary frequency, burning on urination, and lower back pain, The ascending movement of bacteria from the bladder is enhanced with conditions that interfere with the downward flow of urine from the ureters to the bladder or the incomplete emptying of the bladder during urination. These include obstructions such as renal calculi, pregnancy, and reflux of urine from the bladder back into the ureters (vesicoureteral reflux). With appropriate antibiotic therapy and removal of any underlying conditions, acute pyelonephritis can be resolved without permanent damage to the tubules. Urinalysis results are similar to those seen in cystitis, including numerous leukocytes and bacteria with mild proteinuria and hematuria. The additional finding of WBC casts, signifying infection within the tubules, is of primary diagnostic value for both acute and chronic pyelonephritis. Sediments also should be carefully observed for the presence of bacterial casts. Chronic Pyelonephritis As its name implies, chronic pyelonephritis is a serious disorder that can result in permanent damage to the renal tubules and possible progression to chronic renal failure. Congenital urinary structural defects producing reflux nephropathy are the most frequent cause of chronic pyelonephritis. The structural abnormalities may cause reflux between the bladder and ureters or within the renal pelvis, affecting emptying of the collecting ducts. Owing to its congenital origin, chronic pyelonephritis is often diagnosed in children and may not be suspected until tubular damage has become advanced. Urinalysis results are similar to those seen in acute pyelonephritis, particularly in the early stages. As the disease progresses, a variety of granular, waxy, and broad casts are present, accompanied by increased proteinuria and hematuria, and renal concentration is decreased. TECHNICAL TIP The presence of WBC casts is significant for differentiating between cystitis and pyelonephritis. 155 Acute Interstitial Nephritis Acute interstitial nephritis (AIN) is marked by inflammation of the renal interstitium followed by inflammation of the renal tubules. Patients present with a rapid onset of symptoms relating to renal dysfunction, including oliguria, edema, decreased renal concentrating ability, and a possible decrease in the glomerular filtration rate. Fever and the presence of a skin rash are frequent initial symptoms. AIN is primarily associated with an allergic reaction to medications that occurs within the renal interstitium, possibly caused by the medication binding to the interstitial protein. Symptoms tend to develop approximately 2 weeks following administration of medication. Medications commonly associated with AIN include penicillin, methicillin, ampicillin, cephalosporins, sulfonamides, NSAIDs, and thiazide diuretics. Discontinuing the offending medication and administering steroids to control the inflammation frequently results in a return to normal renal function. However, supportive renal dialysis may be required to maintain patients until the inflammation subsides. Urinalysis results include hematuria, possibly macroscopic, mild to moderate proteinuria, numerous WBCs, and WBC casts without bacteria. Differential leukocyte staining for the presence of increased eosinophils may be useful to confirm the diagnosis.10 Laboratory testing and clinical information for the interstitial disorders is summarized in Tables 7–5 and 7–6. Renal Failure Renal failure exists in both acute and chronic forms. As discussed in conjunction with many of the previous disorders, this may be a gradual progression from the original disorder to chronic renal failure or endstage renal disease. The progression to end-stage renal disease is characterized by a marked decrease in the glomerular filtration rate (less than 25 mL/min); steadily rising serum BUN and creatinine values (azotemia); electrolyte imbalance; lack of renal concentrating ability producing an isosthenuric urine; proteinuria; renal glycosuria; and an abundance of granular, waxy, and broad casts, often referred to as a telescoped urine sediment. Acute renal failure (ARF), in contrast to chronic renal failure, exhibits a sudden loss of renal function and is frequently reversible. Primary causes of ARF include a sudden decrease in blood flow to the kidney (prerenal), acute glomerular and tubular disease (renal), and renal calculi or tumor obstructions (postrenal). As can be seen from the variety of causes (Table 7–7), patients may present with many different symptoms relating to the particular disorder involved; however, a decreased glomerular filtration rate, oliguria, edema, and azotemia are general characteristics. Similar to clinical symptoms, urinalysis findings are varied, but because they relate to the primary cause of the ARF, they can be diagnostically valuable. For example, the presence of RTE cells and casts suggests ATN of prerenal origin; RBCs indicate glomerular injury; WBC casts with or without bacteria 3920_Ch07_147-162 23/01/14 10:21 AM Page 156 156 Part Two | Urinalysis Table 7–5 Laboratory Results in Interstitial Disorders Disorder Primary Urinalysis Results Other Significant Tests Cystitis Leukocyturia Bacteriuria Microscopic hematuria Mild proteinuria Increased pH Leukocyturia Bacteriuria WBC casts Bacterial casts Microscopic hematuria Proteinuria Leukocyturia Bacteriuria WBC casts Urine culture Acute pyelonephritis Chronic pyelonephritis Bacterial casts Granular, waxy, broad casts Hematuria Proteinuria Hematuria Proteinuria Leukocyturia WBC casts Acute interstitial nephritis Table 7–6 Urine culture Urine culture BUN Creatinine eGFR Urine eosinophils BUN Creatinine eGFR Clinical Information Associated With Interstitial Disorders Disorder Etiology Clinical Course Cystitis Ascending bacterial infection of the bladder Acute pyelonephritis Infection of the renal tubules and interstitium related to interference of urine flow to the bladder, reflux of urine from the bladder, and untreated cystitis Recurrent infection of the renal tubules and interstitium caused by structural abnormalities affecting the flow of urine Acute onset of urinary frequency and burning resolved with antibiotics Acute onset of urinary frequency, burning, and lower back pain resolved with antibiotics Chronic pyelonephritis Acute interstitial nephritis Allergic inflammation of the renal interstitium in response to certain medications Frequently diagnosed in children; requires correction of the underlying structural defect Possible progression to renal failure Acute onset of renal dysfunction often accompanied by a skin rash Resolves following discontinuation of medication and treatment with corticosteroids 3920_Ch07_147-162 23/01/14 10:21 AM Page 157 Chapter 7 | Renal Disease Table 7–7 Causes of Acute Renal Failure Prerenal Decreased blood pressure/cardiac output Hemorrhage Burns Surgery Septicemia Renal Acute glomerulonephritis Acute tubular necrosis Acute pyelonephritis Acute interstitial nephritis Postrenal Renal calculi Tumors performed chemically, but examination using x-ray crystallography provides a more comprehensive analysis. Approximately 75% of the renal calculi are composed of calcium oxalate or calcium phosphate. Magnesium ammonium phosphate (struvite), uric acid, and cystine are the other primary calculi constituents. Calcium calculi are frequently associated with metabolic calcium and phosphate disorders and occasionally diet. Magnesium ammonium phosphate calculi are frequently accompanied by urinary infections involving urea-splitting bacteria. The urine pH is often higher than 7.0. Uric acid calculi may be associated with increased intake of foods with high purine content and with uromodulin-associated kidney disease. The urine pH is acidic. Most cystine calculi are seen in conjunction with hereditary disorders of cystine metabolism (see Chapter 8). Patient management techniques include maintaining the urine at a pH incompatible with crystallization of the particular chemicals, maintaining adequate hydration to lower chemical concentration, and suggesting possible dietary restrictions. Urine specimens from patients suspected of passing or being in the process of passing renal calculi are frequently received in the laboratory. The presence of microscopic hematuria resulting from irritation to the tissues by the moving calculus is the primary urinalysis finding. indicate interstitial infection or inflammation of renal origin; and postrenal obstruction may show normal- and abnormalappearing urothelial cells possibly associated with malignancy. Renal Lithiasis Renal calculi (kidney stones) may form in the calyces and pelvis of the kidney, ureters, and bladder. In renal lithiasis, the calculi vary in size from barely visible to large, staghorn calculi resembling the shape of the renal pelvis and smooth, round bladder stones with diameters of 2 or more inches. Small calculi may be passed in the urine, subjecting the patient to severe pain radiating from the lower back to the legs. Larger stones cannot be passed and may not be detected until patients develop symptoms of urinary obstruction. Lithotripsy, a procedure using high-energy shock waves, can be used to break stones located in the upper urinary tract into pieces that can then be passed in the urine. Stones can also be removed surgically. Conditions favoring the formation of renal calculi are similar to those favoring formation of urinary crystals, including pH, chemical concentration, and urinary stasis. Numerous correlation studies between the presence of crystalluria and renal calculi formation have been conducted with varying results. The finding of clumps of crystals in freshly voided urine suggests that conditions may be right for calculus formation. However, owing to the difference in conditions that affect the urine within the body and in a specimen container, little importance can be placed on the role of crystals in predicting calculi formation. Increased crystalluria has been noted during the summer months in persons known to form renal calculi.11 Analysis of the chemical composition of renal calculi plays an important role in patient management. Analysis can be 157 Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Forland, M (ed): Nephrology. Medical Examination Publishing, New York, 1983. 2. Couser, WG: Rapidly progressive glomerulonephritis. In Jacobson, HR, et al: Principles and Practice of Nephrology. BC Decker, Philadelphia, 1991. 3. Kallenberg, CG, Mulder, AH, and Tervaert, JW: Antineutrophil cytoplasmic autoantibodies: A still-growing class of autoantibodies in inflammatory disorders. Am J Med 93(6):675–682, 1992. 4. Frasier, LL, and Hoag, KA: Differential diagnosis of Wegener’s granulomatosis from other small vessel vasculitides. LabMed 38(7): 437–439, 2007. 5. Wasserstein, AG: Membranous glomerulonephritis. In Jacobson, HR, et al: Principles and Practice of Nephrology. BC Decker, Philadelphia, 1991. 6. Donadio, JV: Membranoproliferative glomerulonephritis. In Jacobson, HR, et al: Principles and Practice of Nephrology. BC Decker, Philadelphia, 1991. 7. Bricker, NS, and Kirschenbaum, MA: The Kidney: Diagnosis and Management. John Wiley, New York, 1984 8. Sherbotle, JR, and Hayes, JR: Idiopathic nephrotic syndrome: Minimal change disease and focal segmental glomerulosclerosis. In Jacobson, HR, et al: Principles and Practice of Nephrology. BC Decker, Philadelphia, 1991. 9. Bleyer, AJ, Zivna, M, and Kmoch, S: Uromodulin associated kidney disease. Nephron Clin Prac 118(1): 2011. 10. Bennett, WM, Elzinga, LW, and Porter, GA: Tubulointerstitial disease and toxic nephropathy. In Brenner, BM, and Rector, FC: The Kidney: Physiology and Pathophysiology. WB Saunders, Philadelphia, 1991. 11. Hallson, PC, and Rose, GA: Seasonal variations in urinary crystals. Br J Urol 49(4): 277–284, 1977. 3920_Ch07_147-162 23/01/14 10:21 AM Page 158 158 Part Two | Urinalysis Study Questions 1. Most glomerular disorders are caused by: A. Sudden drops in blood pressure B. Immunologic disorders C. Exposure to toxic substances D. Bacterial infections 2. Dysmorphic RBC casts would be a significant finding with all of the following except: A. Goodpasture syndrome B. Acute glomerulonephritis C. Chronic pyelonephritis D. Henoch-Schönlein purpura 3. Occasional episodes of macroscopic hematuria over periods of 20 or more years are seen with: A. Crescentic glomerulonephritis B. IgA nephropathy C. Nephrotic syndrome D. Wegener granulomatosis 4. Antiglomerular basement membrane antibody is seen with: A. Wegener granulomatosis B. IgA nephropathy C. Goodpasture syndrome D. Diabetic nephropathy 5. Antineutrophilic cytoplasmic antibody is diagnostic for: A. IgA nephropathy B. Wegener granulomatosis C. Henoch-Schönlein purpura D. Goodpasture syndrome 6. Respiratory and renal symptoms are associated with all of the following except: A. IgA nephropathy B. Wegener granulomatosis C. Henoch-Schönlein purpura D. Goodpasture syndrome 7. The presence of fatty casts is associated with all of the following except: A. Nephrotic syndrome B. Focal segmental glomerulosclerosis C. Nephrogenic diabetes insipidus D. Minimal change disease 8. The highest levels of proteinuria are seen with: A. Alport syndrome B. Diabetic nephropathy C. IgA nephropathy D. Nephrotic syndrome 9. Ischemia frequently produces: A. Acute renal tubular necrosis B. Minimal change disorder C. Renal glycosuria D. Goodpasture’s syndrome 10. A disorder associated with polyuria and low specific gravity is: A. Renal glucosuria B. Minimal change disease C. Nephrogenic diabetes insipidus D. Focal segmental glomerulosclerosis 11. An inherited disorder producing a generalized defect in tubular reabsorption is: A. Alport syndrome B. Acute interstitial nephritis C. Fanconi syndrome D. Renal glycosuria 12. A teenage boy who develops gout in his big toe and has a high serum uric acid should be monitored for: A. Fanconi syndrome B. Renal calculi C. Uromodulin-associated kidney disease D. Chronic interstitial nephritis 13. The only protein produced by the kidney is: A. Albumin B. Uromodulin C. Uroprotein D. Globulin 14. The presence of renal tubular epithelial cells and casts is an indication of: A. Acute interstitial nephritis B. Chronic glomerulonephritis C. Minimal change disease D. Acute tubular necrosis 3920_Ch07_147-162 23/01/14 10:21 AM Page 159 Chapter 7 | Renal Disease 15. Differentiation between cystitis and pyelonephritis is aided by the presence of: A. WBC casts B. RBC casts C. Bacteria D. Granular casts 16. The presence of WBCs and WBC casts with no bacteria is indicative of: A. Chronic pyelonephritis B. Acute tubular necrosis C. Acute interstitial nephritis D. Both B and C 17. End-stage renal disease is characterized by all of the following except: A. Hypersthenuria B. Isosthenuria C. Azotemia D. Electrolyte imbalance 159 18. Prerenal acute renal failure could be caused by: A. Massive hemorrhage B. Acute tubular necrosis C. Acute interstitial nephritis D. Malignant tumors 19. The most common composition of renal calculi is: A. Calcium oxalate B. Magnesium ammonium phosphate C. Cystine D. Uric acid 20. Urinalysis on a patient with severe back pain being evaluated for renal calculi would be most beneficial if it showed: A. Heavy proteinuria B. Low specific gravity C. Uric acid crystals D. Microscopic hematuria Case Studies and Clinical Situations 1. A 14-year-old boy who has recently recovered from a sore throat develops edema and hematuria. Significant laboratory results include a BUN of 30 mg/dL (normal 8 to 23 mg/dL) and a positive group A streptococcal antibody test. Results of a urinalysis are as follows: Color: Red Ketones: Negative Clarity: Cloudy Blood: Large Sp. gravity: 1.020 Bilirubin: Negative pH: 5.0 Urobilinogen: Normal Protein: 3+ Nitrite: Negative Glucose: Negative Leukocyte: Trace Microscopic: 100 RBCs/hpf—many dysmorphic forms 5–8 WBCs/hpf 0–2 granular casts/lpf 0–1 RBC casts/lpf a. What disorder do these results and history indicate? b. What specific characteristic was present in the organism causing the sore throat? c. What is the significance of the dysmorphic RBCs? d. Are the WBCs significant? Why or why not? e. What is the expected prognosis of this patient? f. If the above urinalysis results were seen in a 5-year-old boy who has developed a red, patchy rash following recovery from a respiratory infection, what disorder would you suspect? 2. B.J. is a seriously ill 40-year-old man with a history of several episodes of macroscopic hematuria in the past 20 years. The episodes were associated with exercise or stress. Until recently the macroscopic hematuria had spontaneously reverted to asymptomatic microscopic hematuria. Significant laboratory results include a BUN of 80 mg/dL (normal 8 to 23 mg/dL), serum creatinine of 4.5 mg/dL (normal 0.6 to 1.2 mg/dL), creatinine clearance of 20 mL/min (normal 107 to 139 mL/min), serum calcium of 8.0 mg/dL (normal 9.2 to 11.0 mg/dL), serum phosphorus of 6.0 mg/dL (normal 2.3 to 4.7 mg/dL), and an elevated level of serum IgA. Results of a routine urinalysis are as follows: Color: Red Ketones: Negative Clarity: Slightly cloudy Blood: Large Sp. gravity: 1.010 Bilirubin: Negative pH: 6.5 Urobilinogen: Normal Protein: 300 mg/dL Nitrite: Negative Glucose: 250 mg/dL Leukocyte: Trace 3920_Ch07_147-162 23/01/14 10:21 AM Page 160 160 Part Two | Urinalysis Microscopic: >100 RBCs/hpf 2–4 hyaline casts/lpf 8–10 WBCs/hpf 1–5 granular casts/lpf 0–2 waxy casts/lpf 0–2 broad waxy a. What specific disease do the patient’s laboratory results and history suggest? b. Which laboratory result is most helpful in diagnosing this disease? c. What additional diagnosis does his current condition suggest? d. What is the significance of the positive result for urine glucose? e. Is the specific gravity significant? Why or why not? f. What is the significance of the waxy casts? 3. A 45-year-old woman is recovering from injuries received in an automobile accident that resulted in her being taken to the emergency department with severe hypotension. She develops massive edema. Significant laboratory results include a BUN of 30 mg/dL (normal 8 to 23 mg/dL), cholesterol of 400 mg/dL (normal 150 to 240 mg/dL), triglycerides of 840 mg/dL (normal 10 to 190 mg/dL), serum protein of 4.5 mg/dL (normal 6.0 to 7.8 mg/dL), albumin of 2.0 mg/dL (normal 3.2 to 4.5 mg/dL), and a total urine protein of 3.8 g/d (normal 100 mg/d). Urinalysis results are as follows: Color: Yellow Ketones: Negative Clarity: Cloudy Blood: Moderate Sp. gravity: 1.015 Bilirubin: Negative pH: 6.0 Urobilinogen: Normal Protein: 4+ Nitrite: Negative Glucose: Negative Leukocyte: Negative Microscopic: 15–20 RBCs/hpf Moderate free fat droplets 0–2 granular casts/lpf 0–5 WBCs/hpf Moderate cholesterol 0–2 fatty casts/lpf crystals 0–2 oval fat bodies/hpf a. What renal disorder do these results suggest? b. How does the patient’s history relate to this disorder? c. What physiologic mechanism accounts for the massive proteinuria? d. What is the relationship of the proteinuria to the edema? e. What mechanism produces the oval fat bodies? 4. A routinely active 4-year-old boy becomes increasingly less active after receiving several preschool immunizations. His pediatrician observes noticeable puffiness around the eyes. A blood test shows normal BUN and creatinine results and markedly decreased total protein and albumin values. Urinalysis results are as follows: Color: Yellow Ketones: Negative Clarity: Hazy Blood: Small Sp. gravity: 1.018 Bilirubin: Negative pH: 6.5 Urobilinogen: Normal Protein: 4+ Nitrite: Negative Glucose: Negative Leukocyte: Negative Microscopic: 10–15 RBCs/hpf 0–1 hyaline casts/lpf 0–4 WBCs/hpf 0–2 granular casts/lpf Moderate fat droplets 0–1 oval fat bodies/hpf a. What disorder do the patient history, physical appearance, and laboratory results suggest? b. What other renal disorders produce similar urinalysis results? c. What is the expected prognosis for this patient? 5. A 32-year-old construction worker experiences respiratory difficulty followed by the appearance of blood-streaked sputum. He delays visiting a physician until symptoms of extreme fatigue and red urine are present. A chest radiograph shows pulmonary infiltration, and sputum culture is negative for pathogens. Blood test results indicate anemia, increased BUN and creatinine, and the presence of antiglomerular basement membrane antibody. Urinalysis results are as follows: Color: Red Ketones: Negative Clarity: Cloudy Blood: Large Sp. gravity: 1.015 Bilirubin: Negative ph: 6.0 Urobilinogen: Normal Protein: 3+ Nitrite: Negative Glucose: Negative Leukocyte: Trace Microscopic: 100 RBCs/hpf 0–3 hyaline casts/lpf 10–15 WBCs/hpf 0–3 granular casts/lpf 0–2 RBCs casts/lpf a. What disorder do the laboratory results suggest? b. How is this disorder affecting the glomerulus? c. If the antiglomerular membrane antibody test is negative, what disorder might be considered? d. What is the diagnostic test for this disorder? e. By what mechanism does this disorder affect the glomerulus? 6. A 25-year-old pregnant woman comes to the outpatient clinic with symptoms of lower back pain, urinary frequency, and a burning sensation when voiding. 3920_Ch07_147-162 23/01/14 10:21 AM Page 161 Chapter 7 | Renal Disease Her pregnancy has been normal up to this time. She is given a sterile container and asked to collect a midstream clean-catch urine specimen. Routine urinalysis results are as follows: Color: Pale yellow Ketones: Negative Clarity: Hazy Blood: Small Sp. gravity: 1.005 Bilirubin: Negative pH: 8.0 Urobilinogen: Normal Protein: Trace Nitrite: Positive Glucose: Negative Leukocyte: 2+ Microscopic: 6–10 RBCs/hpf Heavy bacteria 40–50 WBCs/hpf Moderate squamous epithelial cells a. What is the most probable diagnosis for this patient? b. What is the correlation between the color and the specific gravity? c. What is the significance of the blood and protein tests? d. Is this specimen suitable for the appearance of glitter cells? Explain your answer. e. What other population is at a high risk for developing this condition? f. What disorder might develop if this disorder is not treated? 7. A 10-year-old patient with a history of recurrent UTIs is admitted to the hospital for diagnostic tests. Initial urinalysis results are as follows: Color: Yellow Ketone: Negative Clarity: Cloudy Blood: Small Sp. gravity: 1.025 Bilirubin: Negative pH: 8.0 Urobilinogen: Normal Protein: 2+ Nitrite: Positive Glucose: Negative Leukocyte: 2+ Microscopic: 6–10 RBCs/hpf 0–2 WBC casts/lpf Many bacteria >100 WBCs/hpf 0–1 bacterial casts/lpf with clumps A repeat urinalysis a day later has the following results: Color: Yellow Ketones: Negative clarity: Cloudy Blood: Small Sp. gravity: >1.035 bilirubin: Negative pH: 7.5 Urobilinogen: Normal Protein: 2+ Nitrite: Positive Glucose: Negative Leukocyte: 2+ 161 Microscopic: 6–10 RBCs/hpf Many bacteria 0–2 WBC casts/lpf >100 WBCs/hpf Moderate birefringent, 0–1 bacterial casts/lpf flat crystals a. What diagnostic procedure was performed on the patient that could account for the differences in the two urinalysis results? b. Considering the patient’s age and history, what is the most probable diagnosis? c. What microscopic constituent is most helpful to this diagnosis? d. What is the most probable cause of this disorder? e. How can the presence of the bacterial cast be confirmed? f. What is the most probable source of the crystals present in the sediment? g. Without surgical intervention, what is the patient’s prognosis? 8. A 35-year-old patient being treated for a sinus infection with methicillin develops fever, a skin rash, and edema. Urinalysis results are as follows: Color: Dark yellow Ketones: Negative Clarity: Cloudy Blood: Moderate Sp. gravity: 1.012 Bilirubin: Negative pH: 6.0 Urobilinogen: Normal Protein: 3+ Nitrite: Negative Glucose: Negative Leukocyte: 2+ Microscopic: 20–30 RBCs/hpf 1–2 WBC casts/lpf >100 WBCs/hpf 1–2 granular casts/lpf After receiving the urinalysis report, the physician orders a test for urinary eosinophils. The urinary eosinophil result is 10%. a. Is the urinary eosinophil result normal or abnormal? b. What is the probable diagnosis for this patient? c. Discuss the significance of the increased WBCs and WBC casts in the absence of bacteria. d. How can this condition be corrected? 9. Following surgery to correct a massive hemorrhage, a 55-year-old patient exhibits oliguria and edema. Blood test results indicate increasing azotemia and electrolyte imbalance. The glomerular filtration rate is 20 mL/min. Urinalysis results are as follows: Color: Yellow Ketones: Negative Clarity: Cloudy Blood: Moderate Sp. gravity: 1.010 Bilirubin: Negative 3920_Ch07_147-162 23/01/14 10:21 AM Page 162 162 Part Two | Urinalysis pH: 7.0 Protein: 3+ Glucose: 2+ Microscopic: 50–60 RBCs/hpf 3–6 WBCs/hpf 3–4 RTE cells/hpf a. b. c. d. e. Urobilinogen: Normal Nitrite: Negative Leukocyte: Negative 2–3 granular casts/lpf 2–3 RTE cell casts/lpf 0–1 waxy casts/lpf 0–1 broad granular casts/lpf What diagnosis do the patient’s history and laboratory results suggest? What is the most probable cause of the patient’s disorder? Is this considered to be of prerenal, renal, or postrenal origin? What is the significance of the specific gravity result? What is the significance of the RTE cells? State two possible reasons for the presence of the broad casts. 10. A 40-year-old man develops severe back and abdominal pain after dinner. The pain subsides during the night but recurs in the morning, and he visits his family physician. Results of a complete blood count and an amylase are normal. Results of a routine urinalysis are as follows: Color: Dark yellow Ketones: Negative Clarity: Hazy Blood: Moderate Sp. gravity: 1.030 Bilirubin: Negative pH: 5.0 Urobilinogen: Normal Protein: Trace Nitrite: Negative Glucose: Negative Leukocytes: Negative Microscopic 15–20 RBCs/hpf 0–2 WBCs/hpf Few squamous epithelial cells a. What condition could these urinalysis results and the patient’s symptoms represent? b. What would account for the crenated RBCs? c. Is there a correlation between the urine color and specific gravity and the patient’s symptoms? d. Based on the primary substance that causes this condition, what type of crystals might have been present? e. What changes will the patient be advised to make in his lifestyle to prevent future occurrences? 3920_Ch08_163-179 23/01/14 10:22 AM Page 163 CHAPTER 8 Urine Screening for Metabolic Disorders LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 8-1 Explain abnormal accumulation of metabolites in the urine in terms of overflow and renal disorders. 8-10 State the significance of increased urinary 5-hydroxyindoleacetic acid. 8-2 Discuss the importance of and the MS/MS testing methods for newborn screening. 8-11 Differentiate between cystinuria and cystinosis, including the differences found during analysis of the urine and the disease processes. 8-3 Name the metabolic defect in phenylketonuria, and describe the clinical manifestations it produces. 8-4 State three causes of tyrosyluria. 8-5 Name the abnormal urinary substance present in alkaptonuria, and explain how its presence may be suspected. 8-6 Discuss the appearance and significance of urine that contains melanin. 8-7 Describe a basic laboratory observation that has relevance in maple syrup urine disease. 8-8 Discuss the significance of ketonuria in a newborn. 8-12 Describe the components in the heme synthesis pathway, including the primary specimens used for their analysis, and explain the cause and clinical significance of major porphyrias and the appearance of porphyrins in urine. 8-13 Define mucopolysaccharides, and name three syndromes in which they are involved. 8-14 State the significance of increased uric acid crystals in newborns’ urine. 8-15 Explain the reason for performing tests for urinaryreducing substances on all newborns. 8-9 Differentiate between the presence of urinary indican owing to intestinal disorders and Hartnup disease. KEY TERMS Alkaptonuria Inborn error of metabolism (IEM) Mucopolysaccharidoses Cystinosis Indicanuria Organic acidemias Cystinuria Lesch-Nyhan disease Phenylketonuria (PKU) Galactosuria Maple syrup urine disease (MSUD) Porphyrinuria Hartnup disease Melanuria Tyrosyluria Homocystinuria Melituria 3920_Ch08_163-179 23/01/14 10:22 AM Page 164 164 Part Two | Urinalysis As discussed in previous chapters, many of the abnormal results obtained in the routine urinalysis are related to metabolic rather than renal disease. Urine as an end product of body metabolism may contain additional abnormal substances not tested for by routine urinalysis. Often these substances can be detected or monitored by additional screening tests that can also be performed in the urinalysis laboratory. Positive screening tests can then be followed up with more sophisticated procedures performed in other sections of the laboratory. The need to perform additional tests may be detected by the observations of alert laboratory personnel when performing the routine analysis or from observations of abnormal specimen color and odor by nursing staff and patients (Table 8–1). In other instances, clinical symptoms and family histories are the deciding factors. Overflow Versus Renal Disorders The appearance of abnormal metabolic substances in the urine can be caused by a variety of disorders that can generally be grouped into two categories, termed the overflow type and the Table 8–1 Abnormal Metabolic Constituents or Conditions Detected in the Routine Urinalysis Color Odor Crystals Homogentisic acid Melanin Phenylketonuria Maple syrup urine disease Isovaleric acidemia Cystinuria Cystinosis Homocystinuria Cystine Leucine Indican Porphyrins Table 8–2 Tyrosine LeschNyhan disease renal type. Overflow disorders result from disruption of a normal metabolic pathway that causes increased plasma concentrations of the nonmetabolized substances. These chemicals either override the reabsorption ability of the renal tubules or are not normally reabsorbed from the filtrate because they are present in only minute amounts. Abnormal accumulations of the renal type are caused by malfunctions in the tubular reabsorption mechanism, as discussed in Chapter 7. The most frequently encountered abnormalities are associated with metabolic disturbances that produce urinary overflow of substances involved in protein, fat, and carbohydrate metabolism. This is understandable when one considers the vast number of enzymes used in the metabolic pathways of proteins, fats, and carbohydrates and the fact that their function is essential for complete metabolism. Disruption of enzyme function can be caused by failure to inherit the gene to produce a particular enzyme, referred to as an inborn error of metabolism (IEM),1 or by organ malfunction from disease or toxic reactions. The most frequently encountered abnormal urinary metabolites are summarized in Table 8–2, and their appearance is classified according to functional defect. Table 8–2 also includes substances and conditions that are covered in this chapter. Newborn Screening Tests Many of the urine tests discussed in this chapter traditionally were performed primarily to detect and monitor newborns for IEMs. In recent years the screening of newborns has increased to include more sensitive detection methods and ever-increasing levels of state-mandated tests for IEMs. Many states currently require testing for as many as 30 or more metabolic disorders.2 As discussed later in this chapter, because many of these disorders cause the buildup of unmetabolized toxic food ingredients, it is important that the defects be detected early in life. Levels of these substances are elevated more rapidly in blood than urine. Therefore, blood collected by infant heel puncture is initially tested. Testing for many substances is now performed using tandem mass spectrophotometry Major Disorders of Protein and Carbohydrate Metabolism Associated With Abnormal Urinary Constituents, Classified by Functional Defect Overflow Inherited Metabolic Renal Phenylketonuria Tyrosinemia Alkaptonuria Maple syrup urine disease Organic acidemias Cystinosis Porphyria Mucopolysaccharidoses Galactosemia Lesch-Nyhan disease Infantile tyrosinemia Melanuria Indicanuria 5-Hydroxyindoleacetic acid Porphyria Hartnup disease Cystinuria 3920_Ch08_163-179 23/01/14 10:22 AM Page 165 Chapter 8 | Urine Screening for Metabolic Disorders (MS/MS). MS/MS is capable of screening the infant blood sample for specific substances associated with particular IEMs. Figure 8–1 shows the standard form collected for testing using MS/MS. Methods for specific gene testing are also rapidly being developed. 165 Amino Acid Disorders The amino acid disorders with urinary screening tests include phenylketonuria (PKU), tyrosyluria, alkaptonuria, melanuria, maple syrup urine disease, organic acidemias, indicanuria, cystinuria, and cystinosis. Phenylalanine-Tyrosine Disorders Major inherited disorders include PKU, tyrosyluria, and alkaptonuria. Metabolic defects cause overproduction of melanin. The relationship of these varied disorders is illustrated in Figure 8–2. Phenylketonuria Figure 8–1 Specimen collection form for MS/MS newborn screening test. The most well known of the aminoacidurias, PKU is estimated to occur in 1 of every 10,000 to 20,000 births and, if undetected, results in severe mental retardation. It was first identified in Norway by Ivan Følling in 1934, when a mother with other mentally retarded children reported a peculiar mousy odor to her child’s urine. Analysis of the urine showed increased Normal Metabolism Metabolic Disorders Phenylalanine Phenylketonuria Enzymes Phenylalanine hydroxylase Tyrosine Phenylpyruvic acid Tyrosinemia Type 2 Tyrosyluria p-Hydroxyphenylpyruvic acid p-Hydroxyphenyllactic acid Tyrosinemia Type 3 Alkaptonuria Homogentisic acid Tyrosinemia Type 1b Tyrosinemia Type 1 Tyrosinemia Type 1a Tyrosine aminotransferase Melanin Thyroxine Epinephrine Normal Byproducts p-Hydroxyphenylpyruvic acid p-Hydroxyphenylpyruvate oxidase Homogentisic acid Homogentisic acid oxidase Maleylacetoacetic acid Maleylacetoacetic acid isomerase Fumarylacetoacetic acid Fumarylacetoacetic acid hydrolase Fumaric acid and acetoacetic acid Figure 8–2 Phenylalanine and tyrosine metabolic pathway including the normal pathway (blue), enzymes (yellow), and disorders caused by failure to inherit particular enzymes (green). 3920_Ch08_163-179 23/01/14 10:22 AM Page 166 166 Part Two | Urinalysis amounts of the keto acids, including phenylpyruvate. As shown in Figure 8–2, this occurs when the normal conversion of phenylalanine to tyrosine is disrupted. Interruption of the pathway also produces children with fair complexions—even in dark-skinned families—owing to the decreased production of tyrosine and its pigmentation metabolite melanin. PKU is caused by failure to inherit the gene to produce the enzyme phenylalanine hydroxylase. The gene is inherited as an autosomal recessive trait with no noticeable characteristics or defects exhibited by heterozygous carriers. Fortunately, screening tests are available for early detection of the abnormality, and all states have laws that require the screening of newborns for PKU.2 Once discovered, dietary changes that eliminate phenylalanine, a major constituent of milk, from the infant’s diet can prevent excessive buildup of serum phenylalanine, thereby avoiding damage to the child’s mental capabilities. As the child matures, alternative pathways of phenylalanine metabolism develop, and dietary restrictions can be eased. Many products that contain large amounts of phenylalanine, such as aspartame, now features warning labels for people with PKU. The initial screening for PKU does not come under the auspices of the urinalysis laboratory, because increased blood levels of phenylalanine must, of course, occur before urinary excretion of phenylpyruvic acid, which may take 2 to 6 weeks. State laws require that blood be collected after 24 hours after birth and before the newborn leaves the hospital. The increasing tendency to release newborns from the hospital as early as 24 hours after birth has caused concern about the ability to detect increased phenylalanine levels at that early stage. Studies have shown that in many cases phenylalanine can be detected as early as 4 hours after birth and, if the cutoff level for normal results is lowered from 4 mg/dL to 2 mg/dL, the presence of PKU should be detected. Tests may need to be repeated during an early visit to the pediatrician. More girls than boys escape detection of PKU during early tests because of slower rises in blood phenylalanine levels.1 Urine testing using ferric chloride may be used as a followup test to ensure proper dietary control in previously diagnosed cases and as a means of monitoring the dietary intake of pregnant women known to lack phenylalanine hydroxylase. Urine tests for phenylpyruvic acid are based on the ferric chloride reaction performed by tube test. The addition of ferric chloride to urine containing phenylpyruvic acid produces a permanent blue-green color (see Procedure 8-1). of tyrosine metabolism may result from either inherited or metabolic defects. Also, because two reactions are directly involved in the metabolism of tyrosine, the urine may contain excess tyrosine or its degradation products, p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid. Most frequently seen is a transitory tyrosinemia in premature infants, which is caused by underdevelopment of the liver function required to produce the enzymes necessary to complete the tyrosine metabolism. Acquired severe liver disease also produces tyrosyluria resembling that of the transitory newborn variety and, of course, is a more serious condition. In both instances, rarely seen tyrosine and leucine crystals may be observed during microscopic examination of the urine sediment. Hereditary disorders in which enzymes required in the metabolic pathway are not produced present serious and often fatal conditions that result in both liver and renal tubular disease producing a generalized aminoaciduria. Based on the enzymes affected, the hereditary disorders can be classified into three types, all producing tyrosylemia and tyrosyluria. As shown in Figure 8–2, type 1 is caused by the deficiency of the enzyme fumarylacetoacetate hydrolase (FAH). Type 1 produces a generalized renal tubular disorder and progressive liver failure in infants soon after birth. Type 2 tyrosinemia is caused by lack of the enzyme tyrosine aminotransferase. Persons develop corneal erosion and lesions on the palms, fingers, and soles of the feet believed to be caused by crystallization of tyrosine in the cells. Type 3 tyrosinemia is caused by lack of the enzyme p-hydroxyphenylpyruvic acid dioxygenase. This can result in mental retardation if dietary restrictions of phenylalanine and tyrosine are not implemented. Screening tests using MS/MS are available for tyrosinemia types 1, 2, and 3. See Procedure 8-2 for urine testing for tyrosyluria using nitroso-naphthol. Melanuria The previous discussion focused on the major phenylalaninetyrosine metabolic pathway illustrated in Figure 8–2; however, as also shown in Figure 8–2 and is the case with many amino acids, a second metabolic pathway also exists for tyrosine. This pathway is responsible for the production of Tyrosyluria PROCEDURE 8-2 The accumulation of excess tyrosine in the plasma (tyrosinemia) producing urinary overflow may be due to several causes and is not well categorized. As can be seen in Table 8–2, disorders Nitroso-Naphthol Test for Tyrosine 1. Place five drops of urine in a tube. 2. Add 1 mL of 2.63N nitric acid. 3. Add one drop of 21.5% sodium nitrite. 4. Add 0.1 mL 1-nitroso-2-napthol. 5. Mix. 6. Wait 5 minutes. 7. Observe for an orange-red color, indicating tyrosine metabolites. PROCEDURE 8-1 Ferric Chloride Tube Test 1. Place 1 mL of urine in a tube. 2. Slowly add five drops of 10% ferric chloride. 3. Observe color for a permanent blue-green color. 3920_Ch08_163-179 23/01/14 10:23 AM Page 167 Chapter 8 | Urine Screening for Metabolic Disorders 167 melanin, thyroxine, epinephrine, protein, and tyrosine sulfate. Of these substances, the major concern of the urinalysis laboratory is melanin, the pigment responsible for the dark color of hair, skin, and eyes. Deficient production of melanin results in albinism. Increased urinary melanin darkens the urine. The darkening appears after the urine is exposed to air. Elevated urinary melanin is a serious finding that indicates proliferation of the normal melanin-producing cells (melanocytes), producing a malignant melanoma. These tumors secrete a colorless precursor of melanin, 5,6-dihydroxyindole, which oxidizes to melanogen and then to melanin, producing the characteristic dark urine. PROCEDURE 8-3 Alkaptonuria TECHNICAL TIP It is important to differentiate between the presence of homogentisic acid and melanin when urine is observed to have turned black upon standing. Alkaptonuria was one of the six original inborn errors of metabolism described by Garrod in 1902. The name alkaptonuria was derived from the observation that urine from patients with this condition darkened after becoming alkaline from standing at room temperature. Therefore, the term “alkali lover,” or alkaptonuria, was adopted. This metabolic defect is actually the third major defect in the phenylalanine-tyrosine pathway and occurs from failure to inherit the gene to produce the enzyme homogentisic acid oxidase. Without this enzyme, the phenylalanine-tyrosine pathway cannot proceed to completion, and homogentisic acid accumulates in the blood, tissues, and urine. This condition does not usually manifest itself clinically in early childhood, but observations of brown-stained or black-stained cloth diapers and reddish-stained disposable diapers have been reported.3 In later life, brown pigment becomes deposited in the body tissues (particularly noticeable in the ears). Deposits in the cartilage eventually lead to arthritis. A high percentage of persons with alkaptonuria develop liver and cardiac disorders.4 Homogentisic acid reacts in several of the routinely used screening tests for metabolic disorders, including the ferric chloride test, in which a transient deep blue color is produced in the tube test. A yellow precipitate is produced in the Clinitest, indicating the presence of a reducing substance. Another screening test for urinary homogentisic acid is to add alkali to freshly voided urine and to observe for darkening of the color; however, large amounts of ascorbic acid interfere with this reaction. Paper and thin layer chromatography procedures are available for quantitating homogentisic acid. The silver nitrate test for homogentisic acid is provided in Procedure 8-3. TECHNICAL TIP The appearance of black urine from a patient of any age should be reported to a supervisor. TECHNICAL TIP Melanin may react with sodium nitroferricyanide (acetone reagent strip), producing a red color. Homogentisic Acid Test 1. Place 4 mL of 3% silver nitrate in a tube. 2. Add 0.5 mL of urine. 3. Mix. 4. Add 10% NH4OH by drops. 5. Observe for black color. Branched-Chain Amino Acid Disorders The branched-chain amino acids differ from other amino acids by having a methyl group that branches from the main aliphatic carbon chain (Fig. 8–3 A and B). Two major groups of disorders are associated with errors in the metabolism of the branched-chain amino acids. In one group, accumulation of one or more of the early amino acid degradation products occurs, as is seen in maple syrup urine disease. Disorders in the other group are termed organic acidemias and result in accumulation of organic acids produced further down in the amino acid metabolic pathway. A significant laboratory finding in branched-chain amino acid disorders is the presence of ketonuria in a newborn. Maple Syrup Urine Disease Although maple syrup urine disease (MSUD) is rare, a brief discussion is included in this chapter because the urinalysis laboratory can provide valuable information for the essential early detection of this disease. MSUD is also included in newborn screening profiles using MS/MS. MSUD is caused by an IEM, inherited as an autosomal recessive trait. The amino acids involved are leucine, isoleucine, and valine. The metabolic pathway begins normally, with the transamination of the three amino acids in the liver to the keto acids (α -ketoisovaleric, α -ketoisocaproic, and α -keto-β -methylvaleric). Failure to inherit the gene for the enzyme necessary to produce oxidative decarboxylation of these keto acids results in their accumulation in the blood and urine.1 Newborns with MSUD begin to exhibit clinical symptoms associated with failure to thrive after approximately l week. The presence of the disease may be suspected from these clinical symptoms; however, many other conditions have similar symptoms. Personnel in the urinalysis laboratory or in the nursery may detect the disease by noticing a urine specimen that produces a strong odor resembling maple syrup that is caused by the rapid accumulation of keto acids in the urine. Even though 3920_Ch08_163-179 23/01/14 10:23 AM Page 168 Part Two | Urinalysis 168 Alpha Amino Group H H N H A Alpha Amino Group C O H R Group (Variant) H N C R Carboxyl Group H OH C a report of urine odor is not a part of the routine urinalysis, notifying the physician about this unusual finding can prevent progression to severe mental retardation and even death. Studies have shown that if maple syrup urine disease is detected by the 11th day, dietary regulation and careful monitoring of urinary keto acid concentrations can control the disorder.5 The 2,4-dinitrophenylhydrazine (DNPH) urine screening test for MSUD is provided in Procedure 8-4. Organic Acidemias Generalized symptoms of the organic acidemias include early severe illness, often with vomiting accompanied by metabolic acidosis; hypoglycemia; ketonuria; and increased serum ammonia.6 The three most frequently encountered disorders are isovaleric, propionic, and methylmalonic acidemia. Isovaleric acidemia may be suspected when urine specimens, and sometimes even the patient, possess a characteristic odor of “sweaty feet.” This odor is caused by the accumulation of isovalerylglycine due to a deficiency of isovaleryl coenzyme A in the leucine pathway. The presence of isovaleric, propionic, and methylmalonic acidemias can be detected by newborn screening programs using MS/MS. Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting isoleucine, valine, threonine, and methionine to succinyl coenzyme A. Propionic acid is the immediate precursor to methylmalonic acid in this pathway. Tryptophan Disorders The major concern of the urinalysis laboratory in the metabolism of tryptophan is the increased urinary excretion of the metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA). PROCEDURE 8-4 2,4-Dinitrophenylhydrazine (DNPH) Test for MSUD 1. Place 1 mL of urine in a tube. 2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl. 3. Wait 10 minutes. 4. Observe for yellow turbidity or precipitate. C CH H 3C B O CH3 Leucine Group OH Figure 8–3 α -Alpha amino acid and branched chain amino acid structures. A. Structure of an α -amino acid. B. Structure of the branched chain amino acid leucine. Figure 8–4 shows a simplified diagram of the metabolic pathways by which these substances are produced. Other metabolic pathways of tryptophan are not included because they do not relate directly to the urinalysis laboratory. Indicanuria Under normal conditions, most of the tryptophan that enters the intestine is either reabsorbed for use by the body in producing protein or is converted to indole by intestinal bacteria and excreted in the feces. However, in certain intestinal disorders (including obstruction; the presence of abnormal bacteria; malabsorption syndromes; and Hartnup disease, a rare inherited disorder, increased amounts of tryptophan are converted to indole. The excess indole is then reabsorbed from the intestine into the bloodstream and circulated to the liver, where it is converted to indican and then excreted in the urine. Indican excreted in the urine is colorless until oxidized to the dye indigo blue by exposure to air. Early diagnosis of Hartnup disease is sometimes made when a mother reports a blue staining of her infant’s diapers, referred to as the “blue diaper syndrome.” Except in cases of Hartnup disease, correction of the underlying intestinal disorder returns urinary indican levels to normal. The inherited defect in Hartnup disease affects not only the intestinal reabsorption of tryptophan but also the renal tubular reabsorption of other amino acids, resulting in a generalized aminoaciduria (Fanconi syndrome). The defective renal transport of amino acids does not appear to affect other renal tubular functions. Therefore, with proper dietary supplements, including niacin, people with Hartnup disease have a good prognosis.7 5-Hydroxyindoleacetic Acid As shown in Figure 8–4, a second metabolic pathway of tryptophan is for the production of serotonin used in the stimulation of smooth muscles. Serotonin is produced from tryptophan by the argentaffin cells in the intestine and is carried through the body primarily by the platelets. Normally, the body uses most of the serotonin, and only small amounts of its degradation product, 5-HIAA, are available for excretion in the urine. However, when carcinoid tumors involving the argentaffin (enterochromaffin) cells develop, excess amounts of serotonin are produced, resulting in the elevation of urinary 5-HIAA levels. 3920_Ch08_163-179 23/01/14 10:23 AM Page 169 Chapter 8 | Urine Screening for Metabolic Disorders 169 Tryptophan (normal) Abnormal Abnormal Intestinal disorder Malignant tumors Excess indole 5-Hydroxytryptophan Reabsorbed from intestine into bloodstream Liver Indican Indole Serotonin Feces 5-Hydroxyindoleacetic acid (5-HIAA) Urine Exposure to air Indigo blue Figure 8–4 Tryptophan metabolism. Adding nitrous acid and 1-nitroso-2-naphthol to urine that contains 5-HIAA causes the urine to turn purple to black, depending on the amount of 5-HIAA present (Procedure 8-5). The normal daily excretion of 5-HIAA is 2 to 8 mg, and excretion of greater than 25 mg/24 h can be an indication of argentaffin cell tumors.8 The test can be performed on a random or first morning specimen; however, false-negative results can occur based on the specimen concentration and also because 5-HIAA may not be produced at a constant rate throughout the day. If a 24-hour sample is used, it must be preserved with hydrochloric or boric acid. A plasma method using high-performance liquid chromatography with fluorescence detection is also available. Patients must be given explicit dietary instructions before collecting any sample to be tested for 5-HIAA, because serotonin is a major constituent of foods such as bananas, pineapples, and tomatoes. Medications, including phenothiazines and acetanilids, also interfere with results. Patients should be directed to withhold medications for 72 hours before specimen collection. Cystine Disorders Two distinct disorders of cystine metabolism exhibit renal manifestations. Confusion as to their relationship existed for many years following the discovery of renal calculi consisting PROCEDURE 8-5 Silver Nitroprusside Test 1. Place 1 mL of urine in a tube. 2. Add two drops concentrated NH4OH. 3. Add 0.5 mL 5% silver nitrate. 4. Wait 10 minutes. 5. Add five drops sodium nitroprusside. 6. Observe for purple-black color. of cystine. It is now known that although both disorders are inherited, one is a defect in the renal tubular transport of amino acids (cystinuria) and the other is an IEM (cystinosis). A noticeable odor of sulfur may be present in the urine of people with cystine metabolism disorders. Cystinuria As the name implies, cystinuria is marked by elevated amounts of the amino acid cystine in the urine. The presence of increased urinary cystine is not due to a defect in the metabolism of cystine but, rather, to the inability of the renal tubules to reabsorb cystine filtered by the glomerulus. The demonstration that not only cystine but also lysine, arginine, and ornithine are not reabsorbed has ruled out the possibility of an error in metabolism even though the condition is inherited.9 The disorder has two modes of inheritance: one in which reabsorption of all four amino acids—cystine, lysine, arginine, and ornithine—is affected, and the other in which only cystine and lysine are not reabsorbed. Genetic studies have grouped cystinuria into three types based on the two inherited genes and their heterozygous and homozygous inheritance. In general, persons with any form of inheritance may form renal calculi but the calculi are less common in persons in whom only lysine and cystine are affected.10 Approximately 65% of the people in whom all four amino acids are affected can be expected to produce calculi early in life. Because cystine is much less soluble than the other three amino acids, laboratory screening determinations are based on observing cystine crystals in the sediment of concentrated or first morning specimens. Cystine is also the only amino acid found during the analysis of calculi from these patients. A chemical screening test for urinary cystine can be performed using cyanide-nitroprusside. Reduction of cystine by sodium cyanide followed by the addition of nitroprusside produces a red-purple color in a specimen that contains excess cystine (see Procedure 8-6). False-positive reactions occur in the 3920_Ch08_163-179 23/01/14 10:23 AM Page 170 170 Part Two | Urinalysis PROCEDURE 8-6 Cyanide-Nitroprusside Test for Cystine 1. Place 3 mL of urine in a tube. 2. Add 2 mL sodium cyanide. 3. Wait 10 minutes. 4. Add five drops 5% sodium nitroprusside. 5. Observe for red-purple color. an additional screening test for homocystinuria must be performed by following a positive cyanide-nitroprusside test result with a silver-nitroprusside test, in which only homocystine will react. The use of silver nitrate in place of sodium cyanide reduces homocystine to its nitroprusside-reactive form but does not reduce cystine. Consequently, a positive reaction in the silver-nitroprusside test confirms the presence of homocystinuria. Fresh urine should be used when testing for homocystine (see Procedure 8-7). Porphyrin Disorders presence of ketones and homocystine, and additional tests may have to be performed. Cystinosis Regarded as a genuine IEM, cystinosis can occur in three variations, ranging from a severe fatal disorder developed in infancy to a benign form appearing in adulthood. The disorder has two general categories, termed nephropathic and nonnephropathic. The nephropathic category is subdivided into infantile and lateonset cystinosis. A defect in the lysosomal membranes prevents the release of cystine into the cellular cytoplasm for metabolism. The incomplete metabolism of cystine results in crystalline deposits of cystine in many areas of the body, including the cornea, bone marrow, lymph nodes, and internal organs. A major defect in the renal tubular reabsorption mechanism (Fanconi syndrome) also occurs. The renal tubules, particularly the proximal convoluted tubules, are affected by the cystine deposits that interfere with reabsorption. This is not an inherited disorder of renal tubular reabsorption, as seen in cystinuria. Continued deposition of cystine, if untreated, results in renal failure early in life. In infantile nephropathic cystinosis, there is rapid progression to renal failure. In late-onset nephropathic cystinosis, there is a gradual progression to total renal failure. Renal transplants and the use of cystine-depleting medications to prevent the buildup of cystine in other tissues are extending lives. Nonnephropathic cystinosis is relatively benign but may cause some ocular disorders. Routine laboratory findings in infantile nephropathic cystinosis include polyuria, generalized aminoaciduria, positive Clinitest results for reducing substances, and lack of urinary concentration. Porphyrins are the intermediate compounds in the production of heme. The basic pathway for heme synthesis presented in Figure 8–5 shows the three primary porphyrins (uroporphyrin, coproporphyrin, and protoporphyrin) and the porphyrin precursors (α -aminolevulinic acid [ALA] and porphobilinogen). As can be seen, the synthesis of heme can be blocked at a number of stages. Blockage of a pathway reaction results in the accumulation of the product formed just before the interruption. Detection and identification of this product in the urine, bile, feces, or blood can then aid in determining the cause of a specific disorder. The solubility of the porphyrin compounds varies with their structure. ALA, porphobilinogen, and uroporphyrin are the most soluble and readily appear in the urine. Coproporphyrin is less soluble but is found in the urine, whereas protoporphyrin is not seen in the urine. Fecal analysis has usually been performed to detect coproporphyrin and protoporphyrin. However, to avoid false-positive interference, bile is a more acceptable specimen.11 The Centers for Disease Control and Prevention (CDC) recommends analysis of whole blood for the presence of free erythrocyte protoporphyrin (FEP) as a screening test for lead poisoning. Disorders of porphyrin metabolism are collectively termed porphyrias. They can be inherited or acquired from erythrocytic and hepatic malfunctions or exposure to toxic agents. Common causes of acquired porphyrias include lead poisoning, excessive alcohol intake, iron deficiency, chronic liver disease, and renal disease. Inherited porphyrias are much rarer than acquired porphyrias. They are caused by failure to inherit the gene that produces an enzyme needed in the metabolic pathway. The enzyme deficiency sites for some of the more Homocystinuria Defects in the metabolism of the amino acid methionine produce an increase in homocystine throughout the body. The increased homocystine can result in failure to thrive, cataracts, mental retardation, thromboembolic problems, and death. Early detection of this disorder (homocystinuria) and a change in diet that excludes foods high in methionine can alleviate the metabolic problems. Therefore, screening for homocystine is included in newborn screening programs. Newborn screening tests are performed using MS/MS testing. As mentioned, increased urinary homocystine gives a positive result with the cyanide-nitroprusside test. Therefore, PROCEDURE 8-7 Silver Nitroprusside Test for Homocystine 1. Place 1 mL of urine in a tube. 2. Add two drops concentrated NH4OH. 3. Add 0.5 mL 5% silver nitrate. 4. Wait 10 minutes. 5. Add five drops sodium nitroprusside. 6. Observe for red-purple color. 3920_Ch08_163-179 23/01/14 10:23 AM Page 171 Chapter 8 | Urine Screening for Metabolic Disorders 171 HEME SYNTHESIS Glycine and Succinyl-CoA Aminolevulinic acid synthetase γ-Aminolevulinate (ALA) Aminolevulinic acid synthetase Lead exposure ALA hydratase deficiency porphyria Porphobilinogen Uroporphyrinogen synthase Acute intermittent porphyria Hydroxymethylbilane Uroporphyrinogen cosynthase Congenital erythropoietic porphyria Uroporphyrinogen (UPG) Uroporphyrinogen decarboxylase Porphyria cutanea tarda Coproporphyrinogen (CPG) Coproporphyrinogen oxidase Hereditary coproporphyria Protoporphyrinogen Protoporphyrinogen oxidase Variate porphyria Protoporphyrin IX Ferrochelatase Figure 8–5 Pathway of heme formation, including normal pathway (green), enzymes (orange), and stages affected by the major disorders (yellow) of porphyrin metabolism. common porphyrias are shown in Figure 8–5. The inherited porphyrias are frequently classified by their clinical symptoms, either neurologic/psychiatric or cutaneous photosensitivity or a combination of both (Table 8–3). An indication of the possible presence of porphyrinuria is the observation of a red or port wine color to the urine after exposure to air. The port wine urine color is more prevalent in the erythropoietic porphyrias, and staining of the teeth may also occur. As seen with other inherited disorders, the presence of congenital porphyria is sometimes suspected from a red discoloration of an infant’s diapers. The two screening tests for porphyrinuria use the Ehrlich reaction and fluorescence under ultraviolet light in the 550- to 600-nm range. The Ehrlich reaction can be used only for the Lead exposure Erythropoietic protoporphyria Heme detection of ALA and porphobilinogen. Acetyl acetone must be added to the specimen to convert the ALA to porphobilinogen prior to performing the Ehrlich test. The fluorescent technique must be used for the other porphyrins. The Ehrlich reaction that is now included in the Multistix urobilinogen pad was originally used for all urobilinogen testing. Variations of the Ehrlich reaction include the Watson-Schwartz test for differentiation between the presence of urobilinogen and porphobilinogen (see Procedures 8-8 and 8-9) and the Hoesch test (Procedure 8-10). Testing for the presence of porphobilinogen is most useful when patients exhibit symptoms of an acute attack. This can be done with the Hoesch test. Increased porphobilinogen is associated with acute intermittent porphyria. A negative test 3920_Ch08_163-179 23/01/14 10:23 AM Page 172 172 Part Two | Urinalysis Table 8–3 Common Porphyrias Porphyria Elevated Compound(s) Clinical Symptoms Laboratory Testing Acute intermittent porphyria Porphyria cutanea tarda Congenital erythropoietic porphyria Neurologic/psychiatric Photosensitivity Photosensitivity Urine/Ehrlich reaction Urine fluorescence Urine or feces fluorescence Variegate porphyria ALA Porphobilinogen Uroporphyrin Uroporphyrin Coproporphyrin Coproporphyrin Bile or feces fluorescence Erythropoietic protoporphyria Protoporphyrin Photosensitivity/ neurologic Photosensitivity Lead poisoning ALA Neurologic Protoporphyrin HISTORICAL NOTE Vampires in Old Europe Did you ever wonder how the legend of vampires got started? Think about the previous discussion on the symptoms and inheritance of porphyrias. Photosensitivity→ Avoidance of sunlight Pale coloring→ Anemia caused by heme disorder Port wine− colored urine, red-stained teeth→ Drinking blood Psychiatric symptoms→ Abnormal behavior Inherited disorder→ Familial association, small gene pool Dracula is associated with Transylvania, now Romania. Porphyria was a common disease of early royalty in Europe as a result of intermarriage among the royals of different countries. King George III reportedly died blind, deaf, and mad from porphyria. result is obtained in the presence of lead poisoning unless ALA is first converted to porphobilinogen. Fluorescent screening for the other porphyrins uses their extraction into a mixture of glacial acetic acid and ethyl acetate. The solvent layer is then examined. Negative reactions have a faint blue fluorescence. Positive reactions fluoresce as violet, pink, or red, depending on the concentration of porphyrins. If the presence of interfering substances is suspected, the organic layer can be removed to a separate tube, and 0.5 mL of hydrochloric acid added to the tube. Only porphyrins are extracted into the acid layer, which then produces a bright orange-red fluorescence. The fluorescence method does not distinguish among uroporphyrin, coproporphyrin, and protoporphyrin, but it rules out porphobilinogen and ALA. Identifying specific porphyrins requires additional techniques and the analysis of fecal and erythrocyte samples. Increased protoporphyrin is best measured in whole blood. Blood FEP Bile or feces fluorescence Acetoacetic acid + urine/ Ehrlich reaction Blood FEP Mucopolysaccharide Disorders Mucopolysaccharides, or glycosaminoglycans, are a group of large compounds located primarily in the connective tissue. They consist of a protein core with numerous polysaccharide branches. Inherited disorders in the metabolism of these compounds prevent complete breakdown of the polysaccharide portion of the compounds, resulting in accumulation of the incompletely metabolized polysaccharide portions in the lysosomes of the connective tissue cells and their increased excretion in the urine. The products most frequently found in the urine are dermatan sulfate, keratan sulfate, and heparan sulfate, with the appearance of a particular substance being determined by the specific metabolic error that was inherited.12 Therefore, identification of the specific enzyme deficiency is necessary to establish a specific diagnosis. There are many types of mucopolysaccharidoses, but the best known are Hurler syndrome, Hunter syndrome, and Sanfilippo syndrome. In both Hurler and Hunter syndromes, the skeletal structure is abnormal and there is severe mental retardation; in Hurler syndrome, mucopolysaccharides accumulate in the cornea of the eye. Hunter syndrome is inherited as sex-linked recessive and is rarely seen in females. Without treatment, both syndromes are usually fatal during childhood, whereas in Sanfilippo syndrome, the only abnormality is mental retardation. Bone marrow transplants and gene replacement therapy are the most promising treatments for these disorders. Urinary screening tests for mucopolysaccharides may be requested either as part of a routine battery of tests performed on all newborns or on infants who exhibit symptoms of mental retardation or failure to thrive. The most frequently used screening tests are the acid-albumin and cetyltrimethylammonium bromide (CTAB) turbidity tests and the metachromatic staining spot tests. In both the acid-albumin and the CTAB tests, a thick, white turbidity forms when these reagents are added to urine that contains 3920_Ch08_163-179 23/01/14 10:23 AM Page 173 Chapter 8 | Urine Screening for Metabolic Disorders 173 PROCEDURE 8-8 PROCEDURE 8-9 Watson-Schwartz Differentiation Test The classic test for differentiating between urobilinogen, porphobilinogen, and Ehrlich-reactive compounds is the Watson-Schwartz test. The test is performed as follows: 1. To each tube add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 2. Observe the color of the layers. 3. Interpretation: The addition of chloroform to Tube 1 results in the extraction of urobilinogen into the chloroform (bottom) layer, producing a colorless urine (top) layer, and a red chloroform layer on the bottom. Neither porphobilinogen nor other Ehrlich-reactive compounds are soluble in chloroform. Porphobilinogen is also not soluble in butanol; however, urobilinogen and other Ehrlich-reactive compounds are extracted into butanol. Therefore, the addition of butanol to Tube 2 produces a red (upper) butanol layer if urobilinogen or Ehrlich-reactive compounds are present and a colorless butanol layer if porphobilinogen is present. As shown in the figure, urobilinogen is soluble in both chloroform and butanol, and porphobilinogen is soluble in neither. If both urobilinogen and porphobilinogen are present, both layers appear red. Before reporting the test as positive for both substances, an additional chloroform extraction should be performed on the red urine (upper) layer in Tube 1 to ensure that the red color is not due to excess urobilinogen. Watson-Schwartz Test 1. Label two tubes #1 and #2. 2. To each tube add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 3. Vigorously shake both tubes. 4. Place in a rack for layers to settle. 5. Observe both tubes for red color in the layers. Interpretation: Tube 1 Upper layer = urine; if colorless = porphobilinogen or Ehrlich-reactive compounds. Bottom layer = chloroform; if red = urobilinogen. UR B UR B UR B C UR C UR C UR Urobilinogen Porphobilinogen Ehrlich-reactive UR B UR UR C UR C C Urobilinogen/porphobilinogen Watson-Schwartz reactions. Excess urobilinogen If both layers are red, re-extract the urine layer from tube 1. Place 2 mL of urine layer from Tubes 1 and 2 mL chloroform and 4 mL sodium acetate into a new tube. Repeat procedure. Interpretation: Upper layer—urine colorless Bottom layer—chloroform—red = excess urobilinogen Both layers red = porphobilinogen and urobilinogen Tube 2 Upper layer = butanol If red = urobilinogen or Ehrlich-reactive compounds Bottom layer = urine If colorless = porphobilinogen PROCEDURE 8-10 Hoesch Screening Test for Porphobilinogen The Hoesch test is used for rapid screening or monitoring of urinary porphobilinogen. 1. Two drops of urine are added to approximately 2 mL of Hoesch reagent (Ehrlich reagent dissolved in 6 M HCl). 2. Immediately observed the top of the solution for the appearance of a red color that indicates the presence of porphobilinogen. 3. Shake the tube. Interpretation: When the tube is shaken, the red color is seen throughout the solution. The test detects approximately 2 mg/dL of porphobilinogen, and urobilinogen is inhibited by the highly acidic pH. High concentrations of methyldopa and indican, and highly pigmented urines, may produce falsepositive results. 3920_Ch08_163-179 23/01/14 10:23 AM Page 174 174 Part Two | Urinalysis mucopolysaccharides. Turbidity is usually graded on a scale of 0 to 4 after 30 minutes with acid-albumin and after 5 minutes with CTAB.13 (See Procedure 8-11.) Purine Disorders A disorder of purine metabolism known as Lesch-Nyhan disease that is inherited as a sex-linked recessive results in massive excretion of urinary uric acid crystals. Failure to inherit the gene to produce the enzyme hypoxanthine guanine phosphoribosyltransferase is responsible for the accumulation of uric acid throughout the body. Patients suffer from severe motor defects, mental retardation, a tendency toward selfdestruction, gout, and renal calculi. Development is usually normal for the first 6 to 8 months, and the first sign is often uric acid crystals resembling orange sand in diapers.7 Laboratories should be alert for the presence of increased uric acid crystals in pediatric urine specimens. Galactosuria can be caused by a deficiency in any of three enzymes, galactose-1-phosphate uridyl transferase (GALT), galactokinase, and UDP-galactose-4-epimerase. Of these enzymes, it is GALT deficiency that causes the severe, possibly fatal symptoms associated with galactosemia. Newborn screening protocols currently test for the presence of GALT deficiency. The enzyme is measured in the red blood cells as part of the newborn heel puncture protocol. As a result, people with deficiencies in the other two enzymes may still produce galactosuria but have negative newborn screening tests. Galactose kinase deficiency can result in cataracts in adulthood. UDP-galactose-4-epimerase deficiency may be asymptomatic or produce mild symptoms. Other causes of melituria include lactose, fructose, and pentose. Lactosuria may be seen during pregnancy and lactation. Fructosuria is associated with parenteral feeding and pentosuria with ingestion of large amounts of fruit. Additional tests including chromatography can be used to identify other nonglucose reducing substances. Carbohydrate Disorders The presence of increased urinary sugar (melituria) is most frequently due to an inherited disorder. In fact, pentosuria was one of Garrod’s original six IEMs.14 Fortunately, most meliturias cause no disturbance to body metabolism. However, as discussed in Chapter 5, pediatric urine should be routinely screened for the presence of reducing substances using the Clinitest procedure. The finding of a positive copper reduction test result combined with a negative reagent strip glucose oxidase test result is strongly suggestive of a disorder of carbohydrate metabolism. Of primary concern is the presence of galactosuria, indicating the inability to properly metabolize galactose to glucose. The resulting galactosemia with toxic intermediate metabolic products results in infant failure to thrive, combined with liver disorders, cataracts, and severe mental retardation. Early detection of galactosuria followed by removal of lactose (a disaccharide containing galactose and glucose) from the diet can prevent these symptoms. PROCEDURE 8-11 Cetyltrimethylammonium Bromide (CTAB) Test for Mucopolysaccharides 1. Place 5 mL of urine in a tube. 2. Add 1 mL 5% CTAB in citrate buffer. 3. Read turbidity in 5 minutes. TECHNICAL TIP Lesch-Nyhan disease should not be confused with uromodulin-associated kidney disease, in which the uric acid crystals appear later in life. Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Frimpton, GW: Aminoacidurias due to inherited disorders of metabolism. N Engl J Med 1289:835–901, 1973. 2. National newborn screening and genetics resource center. Web site: http:// www.genes-r-us.uthscsa.edu Accessed January 17, 2007. 3. Nyhan, WL, and Sakati, NO: Diagnostic Recognition of Genetic Disease. Lea & Febiger, Philadelphia, 1987. 4. Stanbury, JB: The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 5. Clow, CL, Reade, TH, and Scriver, CR: Outcome of early and long-term management of classical maple syrup urine disease. Pediatrics 68(6):856–862, 1981. 6. Goodman, SI: Disorders of organic acid metabolism. In Emery, AEH, and Rimoin, DL: Principles and Practice of Medical Genetics. Churchill Livingstone, New York, 1990. 7. Jepson, JB: Hartnup’s disease. In Stanbury, JB, Wyngaarden, JB, and Fredrickson, DS (eds): The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 8. Van Leeuwen, AM, Poelhuis-Leth, DJ, and Bladh, ML: Davis’s Comprehensive Laboratory and Diagnostics Handbook. 4th ed. Philadelphia, FA Davis Company; 2011 9. Nyhan, WL: Abnormalities in Amino Acid Metabolism in Clinical Medicine. Appleton-Century-Crofts, Norwalk, CT., 1984. 10. Dello Strolongo, L, et al: Comparison between SLC3A1 and SLC7A9 cystinuria patients and carriers: A need for a new classification. J Am Soc Nephrol 13:2547–2553, 2002. 11. Nuttall, KL: Porphyrins and disorders of porphyrin metabolism. In Burtis, CA, and Ashwood, ER: Tietz Fundamentals of Clinical Chemistry. WB Saunders, Philadelphia, 1996. 12. McKusick, VA, and Neufeld, EF: The mucopolysaccharide storage diseases. In Stanbury, JB, Wyngaarden, JB, and Fredrickson, DS (eds): The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 13. Kelly, S: Biochemical Methods in Medical Genetics. Charles C. Thomas, Springfield, IL, 1977. 14. Garrod, AE: Inborn Errors of Metabolism. Henry Froude & Hodder & Stoughton, London, 1923. 3920_Ch08_163-179 23/01/14 10:24 AM Page 175 Chapter 8 | Urine Screening for Metabolic Disorders 175 Study Questions 1. All states require newborn screening for PKU for early: A. Modifications of diet B. Administration of antibiotics C. Detection of diabetes D. Initiation of gene therapy 2. All of the following disorders can be detected by newborn screening except: A. Tyrosyluria B. MSUD C. Melanuria D. Galactosemia 3. The best specimen for early newborn screening is a: A. Timed urine specimen B. Blood specimen C. First morning urine specimen D. Fecal specimen 4. Abnormal urine screening tests categorized as an overflow disorder include all of the following except: A. Alkaptonuria B. Galactosemia C. Melanuria D. Cystinuria 5. Which of the following disorders is not associated with the phenylalanine-tyrosine pathway? A. MSUD B. Alkaptonuria C. Albinism D. Tyrosinemia 6. The least serious form of tyrosylemia is: A. Immature liver function B. Type 1 C. Type 2 D. Type 3 7. An overflow disorder of the phenylalanine-tyrosine pathway that would produce a positive reaction with the reagent strip test for ketones is: A. Alkaptonuria B. Melanuria C. MSUD D. Tyrosyluria 8. An overflow disorder that could produce a false-positive reaction with Clinitest procedure is: A. Cystinuria B. Alkaptonuria C. Indicanuria D. Porphyrinuria 9. A urine that turns black after sitting by the sink for several hours could be indicative of: A. Alkaptonuria B. MSUD C. Melanuria D. Both A and C 10. Ketonuria in a newborn is an indication of: A. MSUD B. Isovaleric acidemia C. Methylmalonic acidemia D. All of the above 11. Urine from a newborn with MSUD will have a significant: A. Pale color B. Yellow precipitate C. Milky appearance D. Sweet odor 12. Hartnup disease is a disorder associated with the metabolism of: A. Organic acids B. Tryptophan C. Cystine D. Phenylalanine 13. 5-HIAA is a degradation product of: A. Heme B. Indole C. Serotonin D. Melanin 14. Elevated urinary levels of 5-HIAA are associated with: A. Carcinoid tumors B. Hartnup disease C. Cystinuria D. Platelet disorders 3920_Ch08_163-179 23/01/14 10:24 AM Page 176 176 Part Two | Urinalysis 15. False-positive levels of 5-HIAA can be caused by a diet high in: A. Meat B. Carbohydrates C. Starch D. Bananas 22. Which of the following specimens can be used for porphyrin testing? A. Urine B. Blood C. Feces D. All of the above 16. Place the appropriate letter in front of the following statements. A. Cystinuria B. Cystinosis ____ IEM ____ Inherited disorder of tubular reabsorption ____ Fanconi syndrome ____ Cystine deposits in the cornea ____ Early renal calculi formation 23. The two stages of heme formation affected by lead poisoning are: A. Porphobilinogen and uroporphyrin B. Aminolevulinic acid and porphobilinogen C. Coproporphyrin and protoporphyrin D. Aminolevulinic acid and protoporphyrin 17. Blue diaper syndrome is associated with: A. Lesch-Nyhan syndrome B. Phenylketonuria C. Cystinuria D. Hartnup disease 18. Homocystinuria is caused by failure to metabolize: A. Lysine B. Methionine C. Arginine D. Cystine 19. The Ehrlich reaction will only detect the presence of: A. Uroporphyrin B. Porphobilinogen C. Coproporphyrin D. Protoporphyrin 20. Acetyl acetone is added to the urine before performing the Ehrlich test when checking for: A. Aminolevulinic acid B. Porphobilinogen C. Uroporphyrin D. Coproporphyrin 21. The classic urine color associated with porphyria is: A. Dark yellow B. Indigo blue C. Pink D. Port wine 24. Hurler, Hunter, and Sanfilippo syndromes are hereditary disorders affecting metabolism of: A. Porphyrins B. Purines C. Mucopolysaccharides D. Tryptophan 25. Many uric acid crystals in a pediatric urine specimen may indicate: A. Hurler syndrome B. Lesch-Nyhan disease C. Melituria D. Sanfilippo syndrome 26. Deficiency of the GALT enzyme will produce a: A. Positive Clinitest B. Glycosuria C. Galactosemia D. Both A and C 27. Match the metabolic urine disorders with their classic urine abnormalities. ____ PKU A. Sulfur odor ____ Indicanuria B. Sweaty feet odor ____ Cystinuria C. Orange sand in diaper ____ Alkaptonuria D. Mousy odor ____ Lesch-Nyhan disease E. Black color ____ Isovaleric acidemia F. Blue color 3920_Ch08_163-179 23/01/14 10:24 AM Page 177 Chapter 8 | Urine Screening for Metabolic Disorders 177 Case Studies and Clinical Situations 1. A premature infant develops jaundice. Laboratory tests are negative for hemolytic disease of the newborn, but the infant’s bilirubin level continues to rise. Abnormal urinalysis results include a dark yellow color, positive bilirubin, and needle-shaped crystals seen on microscopic examination. a. What is the most probable cause of the infant’s jaundice? b. Could these same urine findings be associated with an adult? Explain your answer. c. What kinds of crystals are present? Name another type of crystal with a spherical shape that is associated with this condition. d. When blood is drawn from this infant, what precaution should be taken to ensure the integrity of the specimen? 2. A newborn develops severe vomiting and symptoms of metabolic acidosis. Urinalysis results are positive for ketones and negative for glucose and other reducing substances. a. If the urine had an odor of “sweaty feet,” what metabolic disorder would be suspected? b. If the newborn was producing dark brown urine with a sweet odor, what disorder would be suspected? c. Would an MS/MS screen be helpful for the diagnosis? 3. A 13-year-old boy is awakened with severe back and abdominal pain and is taken to the emergency department by his parents. A complete blood count is normal. Family history shows that both his father and uncle are chronic kidney stone formers. Results of a urinalysis are as follows: COLOR: Yellow KETONES: Negative APPEARANCE: Hazy BLOOD: Moderate SP. GRAVITY: 1.025 BILIRUBIN: Negative pH: 6.0 UROBILINOGEN: Normal PROTEIN: Negative NITRITE: Negative GLUCOSE: Negative LEUKOCYTE: Negative Microscopic: >15–20 RBCs/hpf Few squamous epithelial cells 0–3 WBCs/hpf Many cystine crystals a. What condition does the patient’s symptoms represent? b. What is the physiologic abnormality causing this condition? c. If amino acid chromatography was performed on this specimen, what additional amino acids could be present? d. Why are they not present in the microscopic constituents? e. Based on the family history, what genetic disorder should be considered? 4. An 8–month-old boy is admitted to the pediatric unit with a general diagnosis of failure to thrive. The parents have observed slowness in the infant’s development of motor skills. They also mention the occasional appearance of a substance resembling orange sand in the child’s diapers. Urinalysis results are as follows: COLOR: Yellow KETONES: Negative APPEARANCE: Slightly BLOOD: Negative hazy SP. GRAVITY: 1.024 BILIRUBIN: Negative pH: 5.0 UROBILINOGEN: Normal PROTEIN: Negative NITRITE: Negative GLUCOSE: Negative LEUKOCYTE: Negative Microscopic: Many uric acid crystals a. Is the urine pH consistent with the appearance of uric acid crystals? b. Is there any correlation between the urinalysis results and the substance observed in the child’s diapers? Explain your answer. c. What disorder do the patient’s history and the urinalysis results indicate? d. Is the fact that this is a male patient of any significance? Explain your answer. e. Name the enzyme that is missing. 5. Shortly after arriving for the day shift in the urinalysis laboratory, a technician notices that an undiscarded urine has a black color. The previously completed report indicates the color to be yellow. a. Is this observation significant? Explain your answer. b. The original urinalysis report showed the specimen to be positive for ketones. Is this significant? Why or why not? c. If the ketones are negative and the pH is 8.0 is this significant? Why or why not? 6. Bobby Williams, age 8, is admitted through the emergency department with a ruptured appendix. Although surgery is successful, Bobby’s recovery is slow, and the 3920_Ch08_163-179 23/01/14 10:24 AM Page 178 178 Part Two | Urinalysis physicians are concerned about his health prior to the ruptured appendix. Bobby’s mother states that he has always been noticeably underweight despite eating a balanced diet and having strong appetite and that his younger brother exhibits similar characteristics. A note in his chart from the first postoperative day reports that the evening nurse noticed a blue coloration in the urinary catheter bag. a. Is the catheter bag color significant? b. What condition can be suspected from this history? c. What is Bobby’s prognosis? 7. An anemic patient is suspected of having lead poisoning. a. What historical urine test was requested? b. What should be added to the urine before testing? c. What element of heme synthesis would this be testing for? d. Name another substance that can be tested for lead poisoning. e. What element of heme synthesis would this test for? 3920_Ch08_163-179 23/01/14 10:24 AM Page 179 3920_Ch09_180-202 23/01/14 10:29 AM Page 180 PART THREE Other Body Fluids Chapter 9: Cerebrospinal Fluid Chapter 10: Semen Chapter 11: Synovial Fluid Chapter 12: Serous Fluid Chapter 13: Amniotic Fluid Chapter 14: Fecal Analysis Chapter 15: Vaginal Secretions 3920_Ch09_180-202 23/01/14 10:29 AM Page 181 CHAPTER 9 Cerebrospinal Fluid LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 9-1 State the three major functions of cerebrospinal fluid (CSF). 9-2 Distribute CSF specimen tubes numbered 1, 2, 3, and possibly 4 to their appropriate laboratory sections and correctly preserve them. 9-3 Describe the appearance of normal CSF and the causes of abnormally appearing CSF. 9-4 Define xanthochromia and state its significance. 9-5 Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap. 9-6 Calculate CSF total, white blood cell and red blood cell counts when given the number of cells seen, amount of specimen dilution, and the squares counted in the Neubauer chamber. 9-7 Describe the leukocyte content of the CSF in bacterial, viral, tubercular, and fungal meningitis. 9-8 Describe and state the significance of macrophages in the CSF. 9-9 Differentiate between the appearance of normal choroidal cells and malignant cells. 9-10 State the reference values for CSF total protein and name three pathologic conditions that produce an elevated CSF protein. 9-11 Determine whether increased CSF albumin or immunoglobulin is the result of damage to the blood–brain barrier or central nervous system production. 9-12 Discuss the significance of CSF electrophoresis, immunophoresis, and isoelectric focusing findings in multiple sclerosis and the identification of CSF. 9-13 State the reference values for CSF glucose and name the possible pathologic significance of a decreased CSF glucose. 9-14 Discuss the diagnostic value of CSF lactate and glutamine determinations. 9-15 Name the microorganism associated with a positive India ink preparation. 9-16 Discuss the diagnostic value of the bacterial and cryptococcal antigen tests. 9-17 Determine whether a suspected case of meningitis is of bacterial, viral, fungal, or tubercular origin when presented with pertinent laboratory data. 9-18 Describe the role of the Venereal Disease Research Laboratories test and fluorescent treponemal antibody-absorption test for syphilis in CSF testing. KEY TERMS Arachnoid granulations Meningitis Traumatic tap Blood–brain barrier Oligoclonal bands Xanthochromia Choroid plexuses Pleocytosis Meninges Subarachnoid space 3920_Ch09_180-202 23/01/14 10:30 AM Page 182 182 Part Three | Other Body Fluids Cerebrospinal fluid (CSF) is a major fluid of the body. CSF provides a physiologic system to supply nutrients to the nervous tissue, remove metabolic wastes, and produce a mechanical barrier to cushion the brain and spinal cord against trauma. Formation and Physiology The brain and spinal cord are lined by the meninges, which consists of three layers: the dura mater (Latin for “hard mother”), the arachnoid (“spiderweb-like”), and the pia mater (Latin for “gentle mother”). The outer layer is the dura mater that lines the skull and vertebral canal. The arachnoid is a filamentous (spider-like) inner membrane. The pia mater is a thin membrane lining the surfaces of the brain and spinal cord (Fig. 9–1). CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles. In adults, approximately 20 mL of fluid is produced every hour. The fluid flows through the subarachnoid space located between the arachnoid and pia mater (Fig. 9–2). To maintain a volume of 90 to 150 mL in adults and 10 to 60 mL in neonates, the circulating fluid is reabsorbed back into the blood capillaries in the arachnoid granulations/villae at a rate equal to its production. The cells of the arachnoid granulations act as one-way valves that respond to pressure within the central nervous system (CNS) and prevent reflux of the fluid.1 The choroid plexuses are capillary networks that form the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion. Therefore, the chemical composition of the CSF does not resemble an ultrafiltrate of plasma. Capillary walls throughout the body are lined with endothelial cells that are loosely connected to allow passage of soluble nutrients and wastes between the plasma and tissues. In the choroid plexuses, the endothelial cells have very tight-fitting junctures that prevent the passage of many molecules. This tight-fitting structure of the endothelial cells in the choroid plexuses is termed the blood–brain barrier. Maintaining the integrity of the blood–brain barrier is essential to protect the brain from chemicals and other substances circulating in the blood that could harm the brain tissue. In contrast, the junctures also prevent the passage of helpful substances including antibodies and medications. Disruption of the blood–brain barrier by diseases such as meningitis and multiple sclerosis allows leukocytes, proteins, and additional chemicals to enter the CSF. Specimen Collection and Handling CSF is routinely collected by lumbar puncture between the third, fourth, or fifth lumbar vertebra. Although this procedure is not complicated, it does require certain precautions, including measurement of intracranial pressure and careful technique to prevent infection or neural tissue damage. The volume of CSF that can be removed is based on the volume available in the patient (adult vs. neonate) and the opening pressure of the CSF, measured when the needle first enters the subarachnoid space. Elevated pressure requires fluid to be withdrawn slowly, with careful monitoring of the pressure, and may prevent collection of a large volume. Specimens are collected in three sterile tubes, which are labeled 1, 2, and 3 in the order in which they are withdrawn. • Tube 1 is used for chemical and serologic tests because these tests are least affected by blood or bacteria introduced as a result of the tap procedure. • Tube 2 is usually designated for the microbiology laboratory. Central canal Gray matter White matter Spinal nerve roots Dorsal root ganglion Arachnoid granulation Superior sagittal sinus Pia mater Skin Spinal nerve Periosteum Skull Dura mater Arachnoid mater Arachnoid Meninges membrane Pia mater Subarachnoid space Gray matter Subarachnoid space Dura mater Brain White matter tissue A B Figure 9–1 The layers of the meninges. A, the layers of the meninges in the brain. B, the layers of the meninges in the spinal cord. (A, from Scanlon & Sanders, Essentials of Anatomy and Physiology, 6th ed., F. A. Davis Company, Philadelphia, 2011, with permission.) 3920_Ch09_180-202 23/01/14 10:30 AM Page 184 184 Part Three | Other Body Fluids TECHNICAL TIP If only one tube can be collected, it must be tested first by microbiology. TECHNICAL TIP It is not unusual for cell counts requested to be performed on both Tubes 1 and 4 to check for cellular contamination by the puncture Table 9–1 Clinical Significance of CSF Appearance Appearance Cause Crystal clear WBCs Hazy, turbid, milky, cloudy Microorganisms Protein Oily Bloody Xanthochromic Xanthochromia is a term used to describe CSF supernatant that is pink, orange, or yellow. A variety of factors can cause xanthochromia, the most common being the presence of RBC degradation products. Depending on the amount of blood and the length of time it has been present, the color will vary from pink (very slight amount of oxyhemoglobin) to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobin to unconjugated bilirubin). Other causes of xanthochromia include elevated serum bilirubin, presence of the pigment carotene, markedly increased protein concentrations, and melanoma pigment. Xanthochromia that is caused by bilirubin due to immature liver function is also commonly seen in infants, particularly premature infants. The clinical significance of CSF appearance is summarized in Table 9–1. Carotene Protein Melanin Clotted Protein Clotting factors Pellicle Protein Clotting factors Traumatic Collection (Tap) Grossly bloody CSF can be an indication of intracranial hemorrhage, but it may also be due to the puncture of a blood vessel during the spinal tap procedure. Three visual examinations of the collected specimens can usually determine whether the blood is the result of hemorrhage or a traumatic tap. Uneven Blood Distribution Blood from a cerebral hemorrhage will be evenly distributed throughout the three CSF specimen tubes, whereas a traumatic tap will leave the heaviest concentration of blood in tube 1, and gradually diminishing amounts in Tubes 2 and 3. Performing Normal Meningitis Meningitis Disorders affecting blood–brain barrier Production of IgG within the CNS Radiographic contrast media RBCs Hemorrhage Traumatic tap Hemoglobin Old hemorrhage Lysed cells from traumatic tap Bilirubin Figure 9–4 Tubes of CSF. Appearance left to right is normal, xanthochromic, hemolyzed, and cloudy. Major Significance RBC degradation Elevated serum bilirubin level Increased serum levels Disorders affecting blood–brain barrier Meningeal melanosarcoma Disorders affecting blood–brain barrier Introduced by traumatic tap Disorders affecting blood–brain barrier Tubercular meningitis red blood cell (RBC) counts on all three tubes to measure decreasing or constant blood is not always reliable.2 Streaks of blood also may be seen in specimens acquired following a traumatic procedure. Clot Formation Fluid collected from a traumatic tap may form clots owing to the introduction of plasma fibrinogen into the specimen. Bloody CSF caused by intracranial hemorrhage does not contain enough fibrinogen to clot. Diseases in which damage to the blood–brain barrier allows increased filtration of protein 3920_Ch09_180-202 23/01/14 10:30 AM Page 185 Chapter 9 | Cerebrospinal Fluid and coagulation factors also cause clot formation but do not usually produce a bloody fluid. These conditions include meningitis, Froin syndrome, and blocked CSF circulation through the subarachnoid space. A classic web-like pellicle is associated with tubercular meningitis and can be seen after overnight refrigeration of the fluid.3 1 2 3 4 5 6 7 8 9 185 Xanthochromic Supernatant RBCs must usually remain in the CSF for approximately 2 hours before noticeable hemolysis begins; therefore, a xanthochromic supernatant would be the result of blood that has been present longer than that introduced by the traumatic tap. Care should be taken, however, to consider this examination in conjunction with those previously discussed, because a very recent hemorrhage would produce a clear supernatant, and introduction of serum protein from a traumatic tap could also cause the fluid to appear xanthochromic. To examine a bloody fluid for the presence of xanthochromia, the fluid should be centrifuged in a microhematocrit tube and the supernatant examined against a white background. Additional testing for differentiation includes microscopic examination and the D-dimer test. The microscopic finding of macrophages containing ingested RBCs (erythrophagocytosis) or hemosiderin granules indicates intracranial hemorrhage. Detection of the fibrin degradation product D-dimer by latex agglutination immunoassay indicates fibrin formation at a hemorrhage site. Cell Count The cell count that is routinely performed on CSF specimens is the leukocyte (white blood cell [WBC]) count. As discussed previously, the presence and significance of RBCs can usually be ascertained from the appearance of the specimen. Therefore, RBC counts are usually determined only when a traumatic tap has occurred and a correction for leukocytes or protein is desired. The RBC count can be calculated by performing a total cell count and a WBC count and subtracting the WBC count from the total count, if necessary. Any cell count should be performed immediately, because WBCs (particularly granulocytes) and RBCs begin to lyse within 1 hour, and 40% of the leukocytes disintegrate after 2 hours.4 Specimens that cannot be analyzed immediately should be refrigerated. Methodology Normal adult CSF contains 0 to 5 WBCs/µ L. The number is higher in children, and as many as 30 mononuclear cells/µ L can be considered normal in newborns.5 Specimens that contain up to 200 WBCs or 400 RBCs/µ L may appear clear, so it is necessary to examine all specimens microscopically.6 An improved Neubauer counting chamber (Fig. 9–5) is routinely used for performing CSF cell counts. Traditionally, electronic cell counters have not been used for performing CSF cell counts, owing to high background counts and poor reproducibility of low counts. Figure 9–5 Neubauer counting chamber depicting the nine large square counting areas. Automation increases precision, standardization, and faster turnaround time for results. Various automated instruments such as the ADVIA 2120i (Siemens Healthcare Diagnostics Incorporated, Deerfield, IL), Sysmex XE-5000 (Sysmex Corporation, Mundelein, IL), Iris iQ200 with Body Fluids Module (Iris Diagnostics, Chatsworth, CA), and the Beckman Coulter LH780 and UniCel DxH800 (Beckman Coulter, Inc.) are available for CSF cells counts. See Appendix A for more information on automated body fluid analyzers. Calculating CSF Cell Counts The standard Neubauer calculation formula used for blood cell counts is also applied to CSF cell counts to determine the number of cells per microliter. Number of cells counted × dilution = cells/µ L Number of cells counted × volume of 1 square This formula can be used for both diluted and undiluted specimens and offers flexibility in the number and size of the squares counted. Many varied calculations are available, including condensations of the formula to provide single factors by which to multiply the cell count. Keep in mind that the purpose of any calculation is to convert the number of cells counted in a specific amount of fluid to the number of cells that would be present in 1 µ L of fluid. Therefore, a factor can be used only when the dilution and counting area are specific for that factor. The methodology presented in this chapter eliminates the need to correct for the volume counted by counting the four large corner squares (0.4 µ L) and the large center square (0.1 µ L) on each side of the counting chamber.7 3920_Ch09_180-202 23/01/14 10:30 AM Page 186 186 Part Three | Other Body Fluids EXAMPLE Number of cells counted × dilution × 1µ L = cells/µ L 1 µ L (0.1 × 10) (volume counted) Total Cell Count Clear specimens may be counted undiluted, provided no overlapping of cells is seen during the microscopic examination. When dilutions are required, calibrated automatic pipettes, not mouth pipetting, are used. Dilutions for total cell counts are made with normal saline, mixed by inversion, and loaded into the hemocytometer with a Pasteur pipette. Cells are counted in the four corner squares and the center square on both sides of the hemocytometer. As shown in the preceding example, the number of cells counted multiplied by the dilution factor equals the number of cells per microliter. WBC Count Lysis of RBCs must be obtained before performing the WBC count on either diluted or undiluted specimens. Specimens requiring dilution can be diluted in the manner described previously, substituting 3% glacial acetic acid to lyse the RBCs. Adding methylene blue to the diluting fluid stains the WBCs, providing better differentiation between neutrophils and mononuclear cells. To prepare a clear specimen that does not require dilution for counting, place four drops of mixed specimen in a clean tube. Rinse a Pasteur pipette with 3% glacial acetic acid, draining thoroughly, and draw the four drops of CSF into the rinsed pipette. Allow the pipette to sit for 1 minute, mix the solution in the pipette, discard the first drop, and load the hemocytometer. As in the total cell count, WBCs are counted in the four corner squares, and the center square on both sides of the hemocytometer and the number is multiplied by the dilution factor to obtain the number of WBCs per microliter. If a different number of squares is counted, use the standard Neubauer formula to obtain the number of cells per microliter. Quality Control of CSF and Other Body Fluid Cell Counts Liquid commercial controls for spinal fluid RBC and WBC counts are available from several manufacturers. They can be purchased at two levels of concentration. In-house controls can also be prepared. All diluents should be checked biweekly for contamination by examining them in a counting chamber under 400× magnification. Contaminated diluents should be discarded and new solutions prepared. The speed of the cytocentrifuge should be checked monthly with a tachometer, and the timing should be checked with a stopwatch. If nondisposable counting chambers are used, they must be soaked in a bactericidal solution for at least 15 minutes and then thoroughly rinsed with water and cleaned with isopropyl alcohol after each use. Differential Count on a CSF Specimen Identifying the type or types of cells present in the CSF is a valuable diagnostic aid. The differential count should be performed on a stained smear and not from the cells in the counting chamber. Poor visualization of the cells as they appear in the counting chamber has led to the laboratory practice of reporting only the percentage of mononuclear and polynuclear cells present, which can result in overlooking abnormal cells with considerable diagnostic importance. To ensure that the maximum number of cells is available for examination, the specimen should be concentrated before preparing the smear. Methods available for specimen concentration include sedimentation, filtration, centrifugation, and cytocentrifugation. Sedimentation and filtration are not routinely used in the clinical laboratory, although they do produce less cellular distortion. Most laboratories that do not have a cytocentrifuge concentrate specimens with routine centrifugation. The specimen is centrifuged for 5 to 10 minutes, supernatant fluid is removed and saved for additional tests, and slides made from the suspended sediment are allowed to air dry and are stained with Wright’s stain. When the differential count is performed, 100 cells should be counted, classified, and reported in terms of percentage. If the cell count is low and finding 100 cells is not possible, report only the numbers of the cell types seen. Cytocentrifugation A diagrammatic view of the principle of cytocentrifugation is shown in Figure 9–6. Fluid is added to the conical chamber, and as the specimen is centrifuged, cells present in the fluid are forced into a monolayer within a 6-mm diameter circle on the slide. Fluid is absorbed by the filter paper blotter, producing a more concentrated area of cells. As little as 0.1 mL of CSF combined with one drop of 30% albumin produces an adequate cell yield when processed with the cytocentrifuge. Adding albumin increases the cell yield and decreases the cellular distortion frequently seen on cytocentrifuged specimens. Positively charged coated slides to attract cells (Shandon, Inc, Pittsburgh, PA) are also available. Cellular distortion may include cytoplasmic vacuoles, nuclear clefting, prominent nucleoli, indistinct nuclear and cytoplasmic borders, and cellular clumping that resembles malignancy. Cells from both the center and periphery of the slide should be examined because cellular characteristics may vary between areas of the slide. A daily control slide for bacteria should also be prepared using 0.2 mL saline and two drops of the 30% albumin currently being used. The slide is stained and examined if bacteria are seen on a patient’s slide. Table 9–2 presents a cytocentrifuge recovery chart for comparison with chamber counts. The chamber count should 3920_Ch09_180-202 23/01/14 10:30 AM Page 187 Chapter 9 | Cerebrospinal Fluid Stainless steel Cytoclip slide clip 187 Completed assembly Cytoslide™ microscope slide Cytofunnel™ disposable sample chamber with attached filter card In-load position In operation Figure 9–7 Normal lymphocytes. Some cytocentrifuge distortion of cytoplasm (×1000). This cutaway drawing illustrates, at left, the in-load position which shows the sample chamber assembly in a tilted-back position, so that the sample is not absorbed by the filter card. During spinning, centrifugal force tilts the assembly upright and forces the sample to flow toward the microscope slide. Figure 9–6 Cytospin 3 cytocentrifuge specimen processing assembly (Courtesy of Shandon, Inc., Pittsburgh, PA). be repeated if too many cells are seen on the slide, and a new slide should be prepared if not enough cells are seen on the slide.7 CSF Cellular Constituents The cells found in normal CSF are primarily lymphocytes and monocytes (Figs. 9–7 and 9–8). Adults usually have a predominance of lymphocytes to monocytes (70:30), whereas the ratio is essentially reversed in children.5 Improved concentration methods are also showing occasional neutrophils in normal CSF.8 The presence of increased numbers of these normal cells (termed pleocytosis) is considered abnormal, as is the finding of immature leukocytes, eosinophils, plasma cells, macrophages, increased tissue cells, and malignant cells. Table 9–2 Cytocentrifuge Recovery Chart7 Number of WBCs Counted in Chamber 0 1–5 6–10 11–20 20 Number of Cells on Cytocentrifuge Slide 0–40 20–100 60–150 150–250 250 Figure 9–8 Normal lymphocytes and monocytes (×500). When pleocytosis involving neutrophils, lymphocytes, or monocytes is present, the CSF differential count is most frequently associated with its role in providing diagnostic information about the type of microorganism that is causing an infection of the meninges (meningitis). A high CSF WBC count of which the majority of the cells are neutrophils is considered indicative of bacterial meningitis. Likewise, a moderately elevated CSF WBC count with a high percentage of lymphocytes and monocytes suggests meningitis of viral, tubercular, fungal, or parasitic origin. As seen in Table 9–3, many pathologic conditions other than meningitis can be associated with abnormal cells in the CSF. Therefore, because laboratory personnel become so accustomed to finding neutrophils, lymphocytes, and monocytes, they should be careful not to overlook other types of cells. Cell forms differing from those found in blood include macrophages, choroid plexus and ependymal cells, spindleshaped cells, and malignant cells. Neutrophils In addition to bacterial meningitis, increased neutrophils also are seen in the early stages (1 to 2 days) of viral, fungal, tubercular, and parasitic meningitis. Neutrophils may also 3920_Ch09_180-202 23/01/14 10:30 AM Page 188 188 Part Three | Other Body Fluids Table 9–3 Predominant Cells Seen in CSF Type of Cell Major Clinical Significance Microscopic Findings Lymphocytes Normal Viral, tubercular, and fungal meningitis Multiple sclerosis Bacterial meningitis Early cases of viral, tubercular, and fungal meningitis Cerebral hemorrhage Normal Viral, tubercular, and fungal meningitis Multiple sclerosis RBCs in spinal fluid All stages of development may be found Neutrophils Monocytes Macrophages Blast forms Lymphoma cells Plasma cells Ependymal, choroidal, and spindle-shaped cells Malignant cells Acute leukemia Disseminated lymphomas Multiple sclerosis Lymphocyte reactions Diagnostic procedures Metastatic carcinomas Primary central nervous system carcinoma Granules may be less prominent than in blood Cells disintegrate rapidly Found mixed with lymphocytes May contain phagocytized RBCs appearing as empty vacuoles or ghost cells, hemosiderin granules, and hematoidin crystals Lymphoblasts, myeloblasts, or monoblasts Resemble lymphocytes with cleft nuclei Traditional and classic forms seen Reactive lymphs Seen in clusters with distinct nuclei and distinct cell walls Seen in clusters with fusing of cell borders and nuclei contain cytoplasmic vacuoles following cytocentrifugation (Fig. 9–9). Granules are also lost more rapidly in CSF. Neutrophils associated with bacterial meningitis may contain phagocytized bacteria (Figs. 9–10 and 9–11). Although of little clinical significance, neutrophils may be increased following central nervous system (CNS) hemorrhage, repeated lumbar punctures, and injection of medications or radiographic dye. Neutrophils with pyknotic nuclei indicate degenerating cells. They may resemble nucleated red blood cells (NRBCs) but usually have multiple nuclei. When a single nucleus is present they can appear similar to NRBCs (Fig. 9–12). NRBCs are seen as a result of bone marrow contamination during the spinal tap (Figs. 9–13 and 9–14). This is found in approximately 1% of specimens.9 Capillary structures and endothelial cells may be seen following a traumatic tap (Fig. 9–15). Figure 9–9 Neutrophils with cytoplasmic vacuoles resulting from cytocentrifugation (×500). Figure 9–10 Neutrophils with intracellular bacteria (×1000). 3920_Ch09_180-202 23/01/14 10:30 AM Page 189 Chapter 9 | Cerebrospinal Fluid Figure 9–11 Neutrophils with intracellular and extracellular bacteria (×1000). Figure 9–12 Neutrophils with pyknotic nuclei. Notice the cell with a single nucleus in the center (×1000). 189 Figure 9–14 Bone marrow contamination (×1000). Notice the immature RBCs and granulocytes. Figure 9–15 Capillary and tissue fragments from a traumatic tap (×100). conjunction with normal cells (Fig. 9–16). Increased lymphocytes are seen in cases of both asymptomatic HIV infection and AIDS. A moderately elevated WBC count (less than 50 WBCs/µ L) with increased normal and reactive lymphocytes and plasma cells may indicate multiple sclerosis or other degenerative neurologic disorders.10 Figure 9–13 Nucleated RBCs seen with bone marrow contamination (×1000). Lymphocytes and Monocytes A mixture of lymphocytes and monocytes is common in cases of viral, tubercular, and fungal meningitis. Reactive lymphocytes containing increased dark blue cytoplasm and clumped chromatin are frequently present during viral infections in Figure 9–16 Broad spectrum of lymphocytes and monocytes in viral meningitis (×1000). 3920_Ch09_180-202 23/01/14 10:30 AM Page 190 190 Part Three | Other Body Fluids Eosinophils Increased eosinophils are seen in the CSF in association with parasitic infections, fungal infections (primarily Coccidioides immitis), and introduction of foreign material, including medications and shunts, into the CNS (Fig. 9–17). Macrophages The purpose of macrophages in the CSF is to remove cellular debris and foreign objects such as RBCs. Macrophages appear within 2 to 4 hours after RBCs enter the CSF and are frequently seen following repeated taps. They tend to have more cytoplasm than monocytes in the peripheral blood (PB) (Fig. 9–18). The finding of increased macrophages indicates a previous hemorrhage (Fig. 9–19). Further degradation of the phagocytized RBCs results in the appearance of dark blue or black ironcontaining hemosiderin granules (Figs. 9–20 through 9–23). Yellow hematoidin crystals represent further degeneration. They are iron-free, consisting of hemoglobin and unconjugated bilirubin (Figs. 9–24 and 9–25). Figure 9–19 Macrophages showing erythrophagocytosis (×500). Nonpathologically Significant Cells Nonpathologically significant cells are most frequently seen after diagnostic procedures such as pneumoencephalography Figure 9–20 Macrophage with RBC remnants (×500). Figure 9–17 Eosinophils (×1000). Notice cytocentrifuge distortion. Figure 9–21 Macrophage with aggregated hemosiderin granules (×500). Figure 9–18 Macrophages. Notice the large amount of cytoplasm and vacuoles (×500). and in fluid obtained from ventricular taps or during neurosurgery. The cells often appear in clusters and can be distinguished from malignant cells by their uniform appearance. Choroidal cells are from the epithelial lining of the choroid plexus. They are seen singularly and in clumps. Nucleoli are usually absent and nuclei have a uniform appearance (Fig. 9–26). 3920_Ch09_180-202 23/01/14 10:30 AM Page 191 Chapter 9 | Cerebrospinal Fluid 191 Figure 9–22 Macrophage containing hemosiderin stained with Prussian blue (×250). Figure 9–25 Macrophages with hemosiderin and hematoidin (×250). Notice the bright yellow color. Figure 9–23 Macrophage with coarse hemosiderin granules (×500). Figure 9–26 Choroidal cells showing distinct cell borders and nuclear uniformity (×500). Figure 9–24 Macrophage containing hemosiderin and hematoidin crystals (×500). Figure 9–27 Ependymal cells. Notice the nucleoli and less distinct cell borders (×1000). Ependymal cells are from the lining of the ventricles and neural canal. They have less defined cell membranes and are frequently seen in clusters. Nucleoli are often present (Fig. 9–27). Spindle-shaped cells represent lining cells from the arachnoid. They are usually seen in clusters and may be seen with systemic malignancies (Fig. 9–28). Malignant Cells of Hematologic Origin Lymphoblasts, myeloblasts, and monoblasts (Figs. 9–29 to 9–31) in the CSF are frequently seen as a serious complication of acute leukemias. Nucleoli are often more prominent than in blood smears. 3920_Ch09_180-202 23/01/14 10:30 AM Page 192 192 Part Three | Other Body Fluids Figure 9–28 Cluster of spindle-shaped cells (×500). Figure 9–31 Monoblasts and two lymphocytes (×1000). Notice the prominent nucleoli. Figure 9–29 Lymphoblasts from acute lymphocytic leukemia (×500). Figure 9–32 Cleaved and noncleaved lymphoma cells (×1000). Figure 9–30 Myeloblasts from acute myelocytic leukemia (×500). Figure 9–33 Lymphoma cells with nucleoli (×500). Lymphoma cells are also seen in the CSF and indicate dissemination from the lymphoid tissue. They resemble large and small lymphocytes and usually appear in clusters of large, small, or mixed cells based on the classification of the lymphoma. Nuclei may appear cleaved, and prominent nucleoli are present (Figs. 9–32 to 9–34). Malignant Cells of Nonhematologic Origin Metastatic carcinoma cells of nonhematologic origin are primarily from lung, breast, renal, and gastrointestinal malignancies. Cells from primary CNS tumors include astrocytomas, retinoblastomas, and medulloblastomas (Fig. 9–35). They usually appear in clusters and must be distinguished from 3920_Ch09_180-202 23/01/14 10:30 AM Page 193 Chapter 9 | Cerebrospinal Fluid 193 Cerebrospinal Protein Figure 9–34 Burkitt lymphoma. Notice characteristic vacuoles (×500). The most frequently performed chemical test on CSF is the protein determination. Normal CSF contains a very small amount of protein. Reference values for total CSF protein are usually listed as 15 to 45 mg/dL, but are somewhat method dependent, and higher values are found in infants and people over age 40.11 This value is reported in milligrams per deciliter and not grams per deciliter, as are plasma protein concentrations. In general, the CSF contains protein fractions similar to those found in serum; however, the ratio of CSF proteins to serum proteins varies among the fractions. As in serum, albumin makes up most of CSF protein. But in contrast to serum, prealbumin is the second most prevalent fraction in CSF. The alpha globulins include primarily haptoglobin and ceruloplasmin. Transferrin is the major beta globulin present; also, a separate carbohydrate-deficient transferrin fraction, referred to as “tau,” is seen in CSF and not in serum. CSF gamma globulin is primarily immunoglobulin G (IgG), with only a small amount of immunoglobulin A (IgA). Immunoglobulin M (IgM), fibrinogen, and beta lipoprotein are not found in normal CSF.12 Clinical Significance of Elevated Protein Values Figure 9–35 Medulloblastoma (×1000). Notice cellular clustering, nuclear irregularities, and rosette formation. normal clusters of ependymal, choroid plexus, lymphoma, and leukemia cells. Fusing of cell walls and nuclear irregularities and hyperchromatic nucleoli are seen in clusters of malignant cells. Slides containing abnormal cells must be referred to pathology. Chemistry Tests Because CSF is formed by filtration of the plasma, one would expect to find the same low-molecular-weight chemicals in the CSF that are found in the plasma. This is essentially true; however, because the filtration process is selective and the chemical composition is controlled by the blood–brain barrier, reference values for CSF chemicals are not the same as the plasma values. Abnormal values result from alterations in the permeability of the blood–brain barrier or increased production or metabolism by the neural cells in response to a pathologic condition. They seldom have the same diagnostic significance as plasma abnormalities. The clinically important CSF chemicals are few; under certain conditions it may be necessary to measure a larger variety. Elevated total protein values are most frequently seen in pathologic conditions. Abnormally low values are present when fluid is leaking from the CNS. The causes of elevated CSF protein include damage to the blood–brain barrier, immunoglobulin production within the CNS, decreased normal protein clearance from the fluid, and neural tissue degeneration. Meningitis and hemorrhage conditions that damage the blood–brain barrier are the most common causes of elevated CSF protein. Many other neurologic disorders can elevate the CSF protein, and finding an abnormal result on clear fluid with a low cell count is not unusual (Table 9–4). Methodology The two most routinely used techniques for measuring total CSF protein use the principles of turbidity production or dyebinding ability. The turbidity method has been adapted to automated instrumentation in the form of nephelometry. Methods for measuring CSF protein are available for most automated chemistry analyzers. Protein Fractions Routine CSF protein procedures are designed to measure total protein concentration. However, diagnosis of neurologic disorders associated with abnormal CSF protein often requires measurement of the individual protein fractions. Protein that appears in the CSF as a result of damage to the integrity of the blood–brain barrier contains fractions proportional to those in plasma, with albumin present in the highest concentration. Diseases, including multiple sclerosis, that stimulate the immunocompetent cells in the CNS show a higher proportion of IgG. 3920_Ch09_180-202 23/01/14 10:30 AM Page 194 194 Part Three | Other Body Fluids Table 9–4 Clinical Causes of Abnormal CSF Protein Values* Elevated Results • Meningitis • Hemorrhage • Primary CNS tumors • Multiple sclerosis • Guillain-Barré syndrome • Neurosyphilis • Polyneuritis Decreased Results • Myxedema • Cushing disease • Connective tissue disease • Polyneuritis • Diabetes • Uremia • CSF leakage/trauma • Recent puncture • Rapid CSF production • Water intoxication * Reference values for protein are usually 15 to 45 mg/dL, but are method dependent, and higher values are found in infants and people older than 40 years. To accurately determine whether IgG is increased because it is being produced within the CNS or is elevated as the result of a defect in the blood–brain barrier, comparisons between serum and CSF levels of albumin and IgG must be made. Methods include the CSF/serum albumin index to evaluate the integrity of the blood–brain barrier and the CSF IgG index to measure IgG synthesis within the CNS. The CSF/serum albumin index is calculated after determining the concentration of CSF albumin in milligrams per deciliter and the serum concentration in grams per deciliter. The formula used is as follows: CSF/serum albumin index = Electrophoresis and Immunophoretic Techniques The primary purpose for performing CSF protein electrophoresis is to detect oligoclonal bands, which represent inflammation within the CNS. The bands are located in the gamma region of the protein electrophoresis, indicating immunoglobulin production. To ensure that the oligoclonal bands are present as the result of neurologic inflammation, simultaneous serum electrophoresis must be performed. Disorders such as leukemia, lymphoma, and viral infections may produce serum banding, which can appear in the CSF as a result of blood– brain barrier leakage or traumatic introduction of blood into the CSF specimen. Banding representing both systemic and neurologic involvement is seen in the serum and CSF with HIV infection.14 The presence of two or more oligoclonal bands in the CSF that are not present in the serum can be a valuable tool in diagnosing multiple sclerosis, particularly when accompanied by an increased IgG index (Fig. 9–36). Other neurologic disorders including encephalitis, neurosyphilis, Guillain-Barré syndrome, and neoplastic disorders also produce oligoclonal banding that may not be present in the serum. Therefore, the presence of oligoclonal banding must be considered in conjunction with clinical symptoms. Oligoclonal banding remains positive during remission of multiple sclerosis, but disappears in other disorders.10 Low protein levels in the CSF make concentration of the fluid before performing electrophoresis essential for most electrophoretic techniques. Better resolution can be obtained using CSF immunofixation electrophoresis (IFE) and isoelectric CSF albumin (mg/dL) Serum albumin (g/dL) An index value less than 9 represents an intact blood– brain barrier. The index increases relative to the amount of damage to the barrier. Calculation of an IgG index, which is actually a comparison of the CSF/serum albumin index with the CSF/serum IgG index, compensates for any IgG entering the CSF via the blood–brain barrier.13 It is performed by dividing the CSF/serum IgG index by the CSF/serum albumin index as follows: IgG index = CSF IgG (mg/dL)/serum IgG (g/dL) CSF albumin (mg/dL)/serum albumin (g/dL) Normal IgG index values vary slightly among laboratories; however, in general, values greater than 0.70 indicate IgG production within the CNS. Techniques for measuring CSF albumin and globulin have been adapted to automated instrumentation. Figure 9–36 Normal and abnormal oligoclonal banding. (Photograph courtesy of the University of Pittsburgh Department of Pathology, Case Index files, Case 059; http://path.upmc.edu/cases/case59.html, with permission.) 3920_Ch09_180-202 23/01/14 10:30 AM Page 195 Chapter 9 | Cerebrospinal Fluid focusing (IEF) followed by silver staining. Specimen concentration is not required by the more sensitive IEF procedure. These techniques are also the method of choice when determining whether a fluid is actually CSF. CSF can be identified based on the appearance of the previously mentioned extra isoform of tau transferrin that is found only in CSF.15 195 The presence of myelin basic protein (MBP) in the CSF indicates recent destruction of the myelin sheath that protects the axons of the neurons (demyelination). The course of multiple sclerosis can be monitored by measuring the amount of MBP in the CSF.16 MBP may also provide a valuable measure of the effectiveness of current and future treatments. Immunoassay techniques are used for measurement.17 provides more reliable information when the initial diagnosis is difficult. Levels greater than 35 mg/dL are frequently seen with bacterial meningitis, whereas in viral meningitis, lactate levels remain lower than 25 mg/dL. CSF lactate levels remain elevated during initial treatment but fall rapidly when treatment is successful, thus offering a sensitive method for evaluating the effectiveness of antibiotic therapy. Tissue destruction within the CNS owing to oxygen deprivation (hypoxia) increases CSF lactic acid levels. Therefore, elevated CSF lactate is not limited to meningitis and can result from any condition that decreases oxygen flow to the tissues. CSF lactate levels are frequently used to monitor severe head injuries. RBCs contain high concentrations of lactate, and falsely elevated results may be obtained on xanthochromic or hemolyzed fluid.8 CSF Glucose CSF Glutamine Glucose enters the CSF by selective transport across the blood– brain barrier, which results in a reference value that is approximately 60% to 70% that of the plasma glucose. If the plasma glucose is 100 mg/dL, then a reference CSF glucose would be approximately 65 mg/dL. For an accurate evaluation of CSF glucose, a blood glucose test must be run for comparison. The blood glucose should be drawn about 2 hours before the spinal tap to allow time for equilibration between the blood and fluid. CSF glucose is analyzed using the same procedures employed for blood glucose. Specimens should be tested immediately because glycolysis occurs rapidly in CSF. The diagnostic significance of CSF glucose is confined to finding values that are decreased relative to plasma values. Elevated CSF glucose values are always a result of plasma elevations. Low CSF glucose values can be of considerable diagnostic value in determining the causative agents in meningitis. A markedly decreased CSF glucose level accompanied by an increased WBC count and a large percentage of neutrophils indicates bacterial meningitis. If the WBCs are lymphocytes instead of neutrophils, tubercular meningitis is suspected. Likewise, if a normal CSF glucose value is found with an increased number of lymphocytes, the diagnosis would favor viral meningitis. Classic laboratory patterns such as those just described may not be found in all cases of meningitis, but they can be helpful when they appear. Decreased CSF glucose values are caused primarily by alterations in the mechanisms of glucose transport across the blood–brain barrier and by increased use of glucose by the brain cells. The common tendency to associate decreased glucose totally with its use by microorganisms and leukocytes cannot account for the variations in glucose concentrations seen in different types of meningitis and the decreased levels seen in other disorders producing damage to the CNS.18 Glutamine is produced from ammonia and α -ketoglutarate by the brain cells. This process serves to remove the toxic metabolic waste product ammonia from the CNS. The normal concentration of glutamine in the CSF is 8 to 18 mg/dL.19 Elevated levels are associated with liver disorders that result in increased blood and CSF ammonia. Excess ammonia in the CNS increases glutamine synthesis; therefore, determining CSF glutamine provides an indirect test for the presence of excess ammonia in the CSF. Several methods of assaying glutamine are available and are based on the measurement of ammonia liberated from the glutamine. This is preferred over the direct measurement of CSF ammonia because the glutamine concentration remains more stable than the volatile ammonia concentration in the collected specimen. The CSF glutamine level also correlates with clinical symptoms much better than does the blood ammonia.19 As the concentration of ammonia in the CSF increases, the supply of α -ketoglutarate becomes depleted; glutamine can no longer be produced to remove the toxic ammonia, and coma ensues. Some disturbance of consciousness is almost always seen when glutamine levels are more than 35 mg/dL.12 Therefore, the CSF glutamine test is frequently requested for patients with coma of unknown origin. Approximately 75% of children with Reye syndrome have elevated CSF glutamine levels.20 A summary of CSF chemistry tests is presented in Table 9–5. Myelin Basic Protein CSF Lactate CSF lactate levels can be a valuable aid in diagnosing and managing meningitis cases. In bacterial, tubercular, and fungal meningitis, CSF lactate levels greater than 25 mg/dL occurs much more consistently than does decreased glucose and Microbiology Tests The role of the microbiology laboratory in analyzing CSF is to identify the causative agent in meningitis. For positive identification, the microorganism must be recovered from the fluid by growing it on the appropriate culture medium. This can take anywhere from 24 hours in cases of bacterial meningitis to 6 weeks for tubercular meningitis. Consequently, in many instances, the CSF culture is actually a confirmatory rather than a diagnostic procedure. However, the microbiology laboratory does have several methods available to provide information for a preliminary diagnosis. These methods include the Gram 3920_Ch09_180-202 23/01/14 10:30 AM Page 196 196 Part Three | Other Body Fluids Table 9–5 CSF Chemistry Tests Chemical Substance Reference Concentration Value, Normal CSF Significance of Increased Concentration Significance of Decreased Concentration Protein 15 to 45 mg/dL CSF leakage Glucose 60% to 70% of plasma concentration 10 to 24 mg/dL 8 to 18 mg/dL Meningitis Hemorrhage Multiple sclerosis None Lactate Glutamine >35 mg/dL: Bacterial meningitis >35 mg/dL: Some disturbance of consciousness stain, acid-fast stain, India ink preparation, and latex agglutination tests. In Table 9–6, laboratory tests used in the differential diagnosis of meningitis are compared. Gram Stain The Gram stain is routinely performed on CSF from all suspected cases of meningitis, although its value lies in detecting bacterial and fungal organisms. All smears and cultures should be performed on concentrated specimens because often only a few organisms are present at the onset of the disease. The CSF should be centrifuged at 1500 g for 15 minutes, and slides and cultures should be prepared from the sediment.21 Use of the cytocentrifuge provides a highly concentrated specimen for Gram stains. Even when concentrated specimens are used, at least a 10% chance exists that Gram stains and cultures will be negative. Thus, blood cultures should be taken, because the causative organism is often present in both the CSF and the blood.8 A CSF Gram stain is one of the most difficult slides to Table 9–6 Bacterial, tubercular, and fungal meningitis None None interpret because the number of organisms present is usually small, and they can easily be overlooked, resulting in a falsenegative report. Also, false-positive reports can occur if precipitated stain or debris is mistaken for microorganisms. Therefore, considerable care should be taken when interpreting a Gram stain. Organisms most frequently encountered include Streptococcus pneumoniae (gram-positive cocci), Haemophilus influenzae (pleomorphic gram-negative rods), Escherichia coli (gram-negative rods), and Neisseria meningitidis (gram-negative cocci). The gram-positive cocci Streptococcus agalactiae and the gram-positive rods Listeria monocytogenes may be encountered in newborns. Acid-fast or fluorescent antibody stains are not routinely performed on specimens unless tubercular meningitis is suspected. Considering the length of time required to culture mycobacteria, a positive report from this smear is extremely valuable. Specimens from possible cases of fungal meningitis are Gram stained and often have an India ink preparation performed Major Laboratory Results for Differential Diagnosis of Meningitis Bacterial Viral Tubercular Fungal Elevated WBC count Neutrophils present Elevated WBC count Lymphocytes present Marked protein elevation Moderate protein elevation Normal glucose level Elevated WBC count Lymphocytes and monocytes present Moderate to marked protein elevation Decreased glucose level Elevated WBC count Lymphocytes and monocytes present Moderate to marked protein elevation Normal to decreased glucose level Lactate level >25 mg/dL Pellicle formation Lactate level >25 mg/dL Positive India ink with Cryptococcus neoformans Positive immunologic test for C. neoformans Markedly decreased glucose level Lactate level >35 mg/dL Positive Gram stain and bacterial antigen tests Normal lactate level 3920_Ch09_180-202 23/01/14 10:30 AM Page 197 Chapter 9 | Cerebrospinal Fluid on them to detect the presence of thickly encapsulated Cryptococcus neoformans (Fig. 9–37). As one of the more frequently occurring complications of AIDS, cryptococcal meningitis is now commonly encountered in the clinical laboratory. Particular attention should be paid to the Gram stain for the classic starburst pattern produced by Cryptococcus, as this may be seen more often than a positive India ink (Fig. 9–38).22 Latex agglutination tests to detect the presence of C. neoformans antigen in serum and CSF provide a more sensitive method than the India ink preparation. However, immunologic testing results should be confirmed by culture and demonstration of the organisms by India ink, because falsepositive reactions do occur. Interference by rheumatoid factor is the most common cause of false-positive reactions.22 Several commercial kits with pretreatment techniques are available and include incubation with dithiothreitol or pronase and boiling with ethylenediaminetetra-acetic acid.23,24 An enzyme immunoassay technique has been shown to produce fewer false-positive results. The lateral flow assay (LAF) can provide a rapid method for detecting C. neoformans. The procedure utilizes a reagent strip coated with monoclonal antibodies that react with the 197 cryptococcal polysaccharide capsule.25 The complete article and procedures images are available at http:// www.mlo-online.com/ 201303ci. Latex agglutination and enzyme-linked immunosorbent assay (ELISA) methods provide a rapid means for detecting and identifying microorganisms in CSF. Test kits are available to detect Streptococcus group B, H. influenzae Type B, S. pneumoniae, N. meningitidis A, B, C, Y, W135, Mycobacterium tuberculosis, Coccidioides immitis, and E. coli K1 antigens. The bacterial antigen test (BAT) does not appear to be as sensitive to N. meningitidis as it is to the other organisms. The BAT should be used in combination with results from the hematology and clinical chemistry laboratories for diagnosing meningitis.26 The Gram stain is still the recommended method for detecting organisms.27 The amoeba Naegleria fowleri is an opportunistic parasite found in ponds, small lakes, and even chlorinated swimming pools. The Naegleria enters the nasal passages and migrates along the olfactory nerves to invade the brain. Motile trophozoites can be observed microscopically by examining a wet preparation of CSF. Nonmotile trophozoites may be seen on cytocentrifuged stained smears accompanied by increased WBCs and no bacteria. Figure 9–39 shows a Naegleria trophozoite. Notice the elongated form with a tapered posterior.28 Serologic Testing Figure 9–37 India ink preparation of C. neoformans (×400). Notice budding yeast form. (Courtesy of Ann K. Fulenwider, MD.) Figure 9–38 Gram stain of C. neoformans showing starburst pattern (×1000). (Courtesy of Ann K. Fulenwider, MD.) In addition to the serologic procedures performed for identification of microorganisms, serologic testing of the CSF is performed to detect the presence of neurosyphilis. The use of penicillin in the early stages of syphilis has greatly reduced the number of neurosyphilis cases. Consequently, the number of requests for serologic tests for syphilis on CSF is currently low. However, detecting the antibodies associated with syphilis in the CSF still remains a necessary diagnostic procedure. Although many different serologic tests for syphilis are available when testing blood, the procedure recommended by the CDC to diagnose neurosyphilis is the Venereal Disease Research Laboratories (VDRL), even though it is not as sensitive as the fluorescent treponemal antibody-absorption (FTA-ABS) test for syphilis. The rapid plasma regain (RPR) test is not recommended because it is less sensitive than the VDRL. If the FTA-ABS is used, care must be taken to prevent contamination with blood, because the FTA-ABS remains positive in the serum of treated cases of syphilis.29 Methodology for preparing a simulated CSF for student laboratory testing is included in the Instructor’s Guide that accompanies this textbook.30,31 Figure 9–39 Naegleria fowleri trophozoite. (From Leventhal & Cheadle, Medical Parasitology, 6th ed., 2012, FA Davis Company, Philadelphia, with permission.) 3920_Ch09_180-202 23/01/14 10:30 AM Page 198 198 Part Three | Other Body Fluids Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Scanlon,VC and Sanders,T: Essentials of Anatomy and Physiology, 5th ed. FA Davis Company, Philadelphia, 2007. 2. Edlow, JA, and Caplan, LR: Avoiding pitfalls in the diagnosis of subarachnoid hemorrhage. N Engl J Med 342:29–36, 2000. 3. Nagda, KK: Procoagulant activity of cerebrospinal fluid in health and disease. Indian J Med Res 74:107–110, 1981. 4. Chow, G, and Schmidley, JW: Lysis of erythrocytes and leukocytes in traumatic lumbar punctures. Arch Neurol 41:1084–1085, 1984. 5. Seehusen, DA, Reeves, MM, and Fomin, DA: Cerebrospinal fluid analysis. Am Fam Physician 68(6):1103–1108, 2003. 6. Glasser, L: Tapping the wealth of information in CSF. Diagn Med 4(1):23–33, 1981. 7. University of Virginia Health Sciences Center: Clinical Laboratory Procedure Manual. Charlottesville, VA, 1993. 8. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous and Synovial Fluids: A Textbook Atlas. ASCP, Chicago, 1993. 9. Abrams, J, and Schumacher, HR: Bone marrow in cerebrospinal fluid and possible confusion with malignancy. Arch Pathol Lab Med 110:366–369, 1986. 10. Bentz, JS: Laboratory investigation of multiple sclerosis. Lab Med 26(6):393–399, 1995. 11. Biou, D, et al: Cerebrospinal fluid protein concentrations in children: Age-related values in patients without disorders of the central nervous system. Clin Chem 46(3):399–403, 2000. 12. Fishman, RA: Cerebrospinal Fluid in Diseases of the Nervous System, ed 2. WB Saunders, Philadelphia, 1992. 13. Hershey, LA, and Trotter, JL: The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation. Ann Neurol 8(4):426–434, 1980. 14. Grimaldi, LME, et al: Oligoclonal IgG bands in cerebrospinal fluid and serum during asymptomatic human immunodeficiency virus infection. Ann Neurol 24:277–279, 1988. 15. Rouah, E, Rogers, BB, and Buffone, GJ: Transferrin analysis by immunofixation as an aid in the diagnosis of cerebrospinal fluid otorrhea. Arch Pathol Lab Med 111:756–757, 1987. 16. Whitaker, JN: Myelin basic protein in cerebrospinal fluid and other body fluids. Multiple Sclerosis 4(1):16–21, 1998. 17. Okta, M, et al: Evaluation of an enzyme immunoassay for myelin basic protein in CSF. Clin Chem 46:1326–1330, 2000. 18. Menkes, J: The causes of low spinal fluid sugar in bacterial meningitis: Another look. Pediatrics 44(1):1–3, 1969. 19. Hourani, BT, Hamlin, EM, and Reynolds, TB: Cerebrospinal fluid glutamine as a measure of hepatic encephalopathy. Arch Intern Med 127:1033–1036, 1971. 20. Glasgow, AM, and Dhiensiri, K: Improved assay for spinal fluid glutamine and values for children with Reye’s syndrome. Clin Chem 20(6):642–644, 1974. 21. Murray, PR, and Hampton, CM: Recovery of pathogenic bacteria from cerebrospinal fluid. J Clin Microbiol 12:554–557, 1980. 22. Sato, Y, et al: Rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment. J Neurol Sci 164(1):72–75, 1999. 23. Eng, RHK, and Person, A: Serum cryptococcal antigen determination in the presence of rheumatoid factor. J Clin Microbiol 14:700–702, 1981. 24. Stockman, L, and Roberts, GD: Specificity of the latex test for cryptococcal antigen: A rapid simple method for eliminating interference. J Clin Microbiol 17(5):945–947, 1983. 25. Knight, FR: New enzyme immunoassay for detecting cryptococcal antigen. J Clin Pathol 45(9):836–837, 1992. 26. Klausner, JD, Vijayan, T, Chiller,T: Sensitivity and specificity of a new cryptococcal antigen lateral flow assay in serum and cerebrospinal fluid. MLO, March 2013. 27. Wojewoda,C: Bacterial Antigen Testing, The Good, the Not So Bad and the Ugly. NewsPath. Accessed February 14, 2013 28. Leventhal, R and Cheadle, RF: Medical Parasitology, ed.6, FA Davis Company, Philadelphia, 2012. 29. Davis, LE, and Schmitt, JW: Clinical significance of cerebrospinal fluid tests for neurosyphilis. Ann Neurol 25:50–53, 1989. 30. Albright, RE, et al: Issues in cerebrospinal fluid management. Am J Clin Pathol 95(3):397–401, 1991. 31. Lofsness, KG, and Jensen, TL: The preparation of simulated spinal fluid for teaching purposes. Am J Med Technology 49(7):493–496, 1983. Study Questions 1. The functions of the CSF include all of the following except: A. Removing metabolic wastes B. Producing an ultrafiltrate of plasma C. Supplying nutrients to the CNS D. Protecting the brain and spinal cord 2. The CSF flows through the: A. Choroid plexus B. Pia mater C. Arachnoid space D. Dura mater 3. Substances present in the CSF are controlled by the: A. Arachnoid granulations B. Blood–brain barrier C. Presence of one-way valves D. Blood–CSF barrier 4. What department is the CSF tube labeled 3 routinely sent to? A. Hematology B. Chemistry C. Microbiology D. Serology 3920_Ch09_180-202 23/01/14 10:30 AM Page 199 Chapter 9 | Cerebrospinal Fluid 5. The CSF tube that should be kept at room temperature is: A. Tube 1 B. Tube 2 C. Tube 3 D. Tube 4 6. Place the appropriate letter in front of the statement that best describes CSF specimens in these two conditions: A. Traumatic tap B. Intracranial hemorrhage ____ Even distribution of blood in all tubes ____ Xanthochromic supernatant ____ Concentration of blood in tube 1 is greater than in tube 3 ____ Specimen contains clots 7. The presence of xanthochromia can be caused by all of the following except: A. Immature liver function B. RBC degradation C. A recent hemorrhage D. Elevated CSF protein 8. A web-like pellicle in a refrigerated CSF specimen indicates: A. Tubercular meningitis B. Multiple sclerosis C. Primary CNS malignancy D. Viral meningitis 9. Given the following information, calculate the CSF WBC count: cells counted, 80; dilution, 1:10; large Neubauer squares counted, 10. A. 8 B. 80 C. 800 D. 8000 10. A CSF WBC count is diluted with: A. Distilled water B. Normal saline C. Acetic acid D. Hypotonic saline 11. A total CSF cell count on a clear fluid should be: A. Reported as normal B. Not reported C. Diluted with normal saline D. Counted undiluted 199 12. The purpose of adding albumin to CSF before cytocentrifugation is to: A. Increase the cell yield B. Decrease the cellular distortion C. Improve the cellular staining D. Both A and B 13. The primary concern when pleocytosis of neutrophils and lymphocytes is found in the CSF is: A. Meningitis B. CNS malignancy C. Multiple sclerosis D. Hemorrhage 14. Neutrophils with pyknotic nuclei may be mistaken for: A. Lymphocytes B. Nucleated RBCs C. Malignant cells D. Spindle-shaped cells 15. The presence of which of the following cells is increased in a parasitic infection? A. Neutrophils B. Macrophages C. Eosinophils D. Lymphocytes 16. Macrophages appear in the CSF after: A. Hemorrhage B. Repeated spinal taps C. Diagnostic procedures D. All of the above 17. Nucleated RBCs are seen in the CSF as a result of: A. Elevated blood RBCs B. Treatment of anemia C. Severe hemorrhage D. Bone marrow contamination 18. After a CNS diagnostic procedure, which of the following might be seen in the CSF? A. Choroidal cells B. Ependymal cells C. Spindle-shaped cells D. All of the above 3920_Ch09_180-202 23/01/14 10:30 AM Page 200 200 Part Three | Other Body Fluids 19. Hemosiderin granules and hematoidin crystals are seen in: A. Lymphocytes B. Macrophages C. Ependymal cells D. Neutrophils 20. Myeloblasts are seen in the CSF: A. In bacterial infections B. In conjunction with CNS malignancy C. After cerebral hemorrhage D. As a complication of acute leukemia 21. Cells resembling large and small lymphocytes with cleaved nuclei represent: A. Lymphoma cells B. Choroid cells C. Melanoma cells D. Medulloblastoma cells 22. The reference range for CSF protein is: A. 6 to 8 g/dL B. 15 to 45 g/dL C. 6 to 8 mg/dL D. 15 to 45 mg/dL 23. CSF can be differentiated from serum by the presence of: A. Albumin B. Globulin C. Prealbumin D. Tau transferrin 24. In serum, the second most prevalent protein is IgG; in CSF, the second most prevalent protein is: A. Transferrin B. Prealbumin C. IgA D. Ceruloplasmin 25. Elevated CSF protein values can be caused by all of the following except: A. Meningitis B. Multiple sclerosis C. Fluid leakage D. CNS malignancy 26. The integrity of the blood–brain barrier is measured using the: A. CSF/serum albumin index B. CSF/serum globulin ratio C. CSF albumin index D. CSF IgG index 27. Given the following results, calculate the IgG index: CSF IgG, 50 mg/dL; serum IgG, 2 g/dL; CSF albumin, 70 mg/dL; serum albumin, 5 g/dL. A. 0.6 B. 6.0 C. 1.8 D. 2.8 28. The CSF IgG index calculated in Study Question 27 indicates: A. Synthesis of IgG in the CNS B. Damage to the blood–brain barrier C. Cerebral hemorrhage D. Lymphoma infiltration 29. The finding of oligoclonal bands in the CSF and not in the serum is seen with: A. Multiple myeloma B. CNS malignancy C. Multiple sclerosis D. Viral infections 30. A CSF glucose of 15 mg/dL, WBC count of 5000, 90% neutrophils, and protein of 80 mg/dL suggests: A. Fungal meningitis B. Viral meningitis C. Tubercular meningitis D. Bacterial meningitis 31. A patient with a blood glucose of 120 mg/dL would have a normal CSF glucose of: A. 20 mg/dL B. 60 mg/dL C. 80 mg/dL D. 120 mg/dL 32. CSF lactate will be more consistently decreased in: A. Bacterial meningitis B. Viral meningitis C. Fungal meningitis D. Tubercular meningitis 33. Measurement of which of the following can be replaced by CSF glutamine analysis in children with Reye syndrome? A. Ammonia B. Lactate C. Glucose D. α -Ketoglutarate 3920_Ch09_180-202 23/01/14 10:30 AM Page 201 Chapter 9 | Cerebrospinal Fluid 34. Before performing a Gram stain on CSF, the specimen must be: A. Filtered B. Warmed to 37°C C. Centrifuged D. Mixed 201 36. The test of choice to detect neurosyphilis is the: A. RPR B. VDRL C. FAB D. FTA-ABS 35. All of the following statements are true about cryptococcal meningitis except: A. An India ink preparation is positive B. A starburst pattern is seen on Gram stain C. The WBC count is over 2000 D. A confirmatory immunology test is available Case Studies and Clinical Situations 1. Three tubes of CSF containing evenly distributed visible blood are drawn from a 75-year-old disoriented patient and delivered to the laboratory. Initial test results are as follows: WBC count: 250 µ L Protein: 150 mg/dL Glucose: 70 mg/dL Gram stain: No organisms seen Differential: Neutrophils, 68%; monocytes, 3%; lymphocytes, 28%; eosinophils, 1% Many macrophages containing ingested RBCs a. What is the most probable condition indicated by these results? State two reasons for your answer. b. Are the elevated WBC count and protein significant? Explain your answer. c. Are the percentages of the cells in the differential of any significance? Explain your answer. d. What two other structures besides RBCs might be contained in the macrophages? e. If the blood was unevenly distributed and nucleated RBCs and capillary structures were seen instead of macrophages, what would this indicate? 2. A patient with AIDS is hospitalized with symptoms of high fever and rigidity of the neck. Routine laboratory tests on the CSF show a WBC count of 100/µ L with a predominance of lymphocytes and monocytes, glucose of 55 mg/dL (plasma: 85 mg/dL), and a protein of 70 mg/dL. The Gram stain shows a questionable starburst pattern. a. What additional microscopic examination should be performed? b. If the test is positive, what is the patient’s diagnosis? c. If the results of the test are questionable, what additional testing can be performed? d. What could cause a false-positive reaction in this test? e. If the tests named in a and c are negative, the glucose level is 35 mg/dL, and a pellicle is observed in the fluid, what additional testing should be performed? f. If CSF and serum IEF was performed on this patient, what unusual findings might be present? 3. A 35-year-old woman is admitted to the hospital with symptoms of intermittent blurred vision, weakness, and loss of sensation in her legs. A lumbar puncture is performed with the following results: Appearance: Colorless, clear WBC count: 35 cells/µ L (90% lymphocytes) Glucose: 60 mg/dL (plasma: 100 mg/dL) Protein: 60 mg/dL (serum: 8 g/dL) Albumin: 40 mg/dL (serum: 6 g/dL) IgG globulin: 20 mg/dL (serum: 2 g/dL) a. Name and perform the calculation to determine the integrity of the patient’s blood–brain barrier. b. Does the patient have an intact barrier? c. Name and perform the calculation used to determine if IgG is being synthesized within the CNS. d. What does this result indicate? e. Considering the patient’s clinical symptoms and the calculation results, what diagnosis is suggested? f. If immunofixation electrophoresis is performed on the patient’s serum and CSF, what findings would be expected? g. What substance in the CSF can be measured to monitor this patient? 3920_Ch09_180-202 23/01/14 10:30 AM Page 202 202 Part Three | Other Body Fluids 4. Mary Howard, age 5, is admitted to the pediatrics ward with a temperature of 105%F, lethargy, and cervical rigidity. A lumbar spinal tap is performed, and three tubes of cloudy CSF are delivered to the laboratory. Preliminary test results are as follows: Appearance: Cloudy WBC count: 800 cells/µ L Differential: 80% lymphocytes, 15% monocytes, 5% neutrophils Protein: 65 mg/dL Glucose: 70 mg/dL Gram stain: No organisms seen a. From these results, what preliminary diagnosis could the physician consider? b. Is the Gram stain result of particular significance? Why or why not? c. Are the lymphocytes significant? Why or why not? d. Would a CSF lactate test be of any value for the diagnosis? Why or why not? 5. State possible technical errors that could result in the following discrepancies: a. An unusual number of Gram stains reported as grampositive cocci fail to be confirmed by positive cultures. b. A physician complains that CSF differentials are being reported only as polynuclear and mononuclear cells. c. Bacteria observed on the cytospin differential cannot be confirmed by Gram stain or culture. d. The majority of CSF specimens sent to the laboratory from the neurology clinic have glucose readings less than 50% of the corresponding blood glucose results performed in the clinic. 3920_Ch10_203-216 23/01/14 10:31 AM Page 203 CHAPTER 10 Semen LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 10-1 State the structures involved in sperm production and their function. 10-10 Describe the appearance of normal sperm, including structures and their functions. 10-2 Describe the four components of semen with regard to source and function. 10-11 Differentiate between routine and strict criteria for evaluating sperm morphology. 10-3 Explain the procedures for collecting and handling semen specimens. 10-12 Given an abnormal result in a routine semen analysis, determine additional tests that might be performed. 10-4 Describe the normal appearance of semen and three abnormalities in appearance. 10-13 Describe the two methods routinely used to detect antisperm antibodies. 10-5 State two possible causes of low semen volume. 10-14 List two methods for identifying a questionable fluid as semen. 10-6 Discuss the significance of semen liquefaction and viscosity. 10-7 Calculate a sperm concentration and count when provided with the number of sperm counted, the dilution, the area of the counting chamber used, and the ejaculate volume. 10-8 Define round cells, and explain their significance. 10-15 State the World Health Organization reference values for routine and follow-up semen analysis. 10-16 Discuss the types and significance of sperm function tests. 10-17 Describe methods of quality control appropriate for semen analysis. 10-9 State the two parameters considered when evaluating sperm motility. KEY TERMS Acrosomal cap Prostate gland Spermatozoa Andrology Semen Vasectomy Bulbourethral gland Seminal vesicles Viscosity Epididymis Seminiferous tubules Liquefaction Spermatids 3920_Ch10_203-216 23/01/14 10:31 AM Page 204 Part Three | Other Body Fluids 204 Advances in the field of andrology and assisted reproductive technology (ART), and increased concern over fertility, particularly by couples choosing to have children later in life, have resulted in increased emphasis on semen analysis. Patients with abnormal results on the routine semen analysis performed in the clinical laboratory often are referred to specialized andrology laboratories for further testing to determine the need for in vitro fertilization (IVF). Clinical laboratory personnel may also be employed in andrology laboratories and perform both routine and specialized testing. In addition to fertility testing, the clinical laboratory performs postvasectomy semen analysis and forensic analyses to determine the presence of semen. Physiology Semen is composed of four fractions that are contributed by the testes, epididymis, seminal vesicles, prostate gland, and bulbourethral glands (Fig. 10–1). Each fraction differs in its composition, and the mixing of all four fractions during ejaculation is essential for the production of a normal semen specimen (Table 10–1). The testes are paired glands in the scrotum that contain the seminiferous tubules for the secretion of sperm. The external location of the scrotum contributes to a lower scrotum temperature that is optimal for sperm development. Germ cells Table 10–1 Semen Composition Spermatozoa Seminal fluid Prostate fluid Bulbourethral glands 5% 60% to 70% 20% to 30% 5% for the production of spermatozoa are located in the epithelial cells of the seminiferous tubules. Specialized Sertoli cells provide support and nutrients for the germ cells as they undergo mitosis and meiosis (spermatogenesis). When spermatogenesis is complete, the immature sperm (nonmotile) enter the epididymis. In the epididymis, the sperm mature and develop flagella. The entire process takes approximately 90 days. The sperm remain stored in the epididymis until ejaculation, at which time they are propelled through the ductus deferens (vas deferens) to the ejaculatory ducts. The ejaculatory ducts receive both the sperm from the ductus deferens and fluid from the seminal vesicles. The seminal vesicles produce most of the fluid present in semen (60% to 70%), and this fluid is the transport medium for the sperm. The fluid contains a high concentration of fructose and flavin. Spermatozoa metabolize the fructose for the energy needed for the flagella to propel them through the female reproductive Urinary bladder Seminal vesicle Rectum Vas deferens Ejaculatory duct Urethra Prostate gland Penis Glans penis Epididymis Testis Scrotum Ureter Vas deferens Prostate gland Anus Bulbourethral gland Urinary bladder Seminal vesicle Bulbourethral gland Epididymis Testis Penis Urethra Figure 10–1 The male genitalia. Top, sagittal view; bottom, anterior view. 3920_Ch10_203-216 23/01/14 10:31 AM Page 205 Chapter 10 | Semen tract. In the absence of fructose, sperm do not display motility in the semen analysis. Flavin is responsible for the gray appearance of semen. Various proteins secreted by the seminal vesicles are involved in the coagulation of the ejaculate. The muscular prostate gland, located just below the bladder, surrounds the upper urethra and aids in propelling the sperm through the urethra by contractions during ejaculation. Approximately 20% to 30% of the semen volume is acidic fluid produced by the prostate gland. The milky acidic fluid contains high concentrations of acid phosphatase, citric acid, zinc, and proteolytic enzymes responsible for both the coagulation and liquefaction of the semen following ejaculation. The bulbourethral glands, located below the prostate, contribute about 5% of the fluid volume in the form of a thick, alkaline mucus that helps to neutralize acidity from the prostate secretions and the vagina. It is important for semen to be alkaline to neutralize the vaginal acidity present as a result of normal bacterial vaginal flora. Without this neutralization, sperm motility would be diminished. Specimen Collection The variety in the composition of the semen fractions makes proper collection of a complete specimen essential for accurate evaluation of male fertility. Most of the sperm are contained in the first portion of the ejaculate, making complete collection essential for accurate testing of both fertility and postvasectomy specimens. When a part of the first portion of the ejaculate is missing, the sperm count will be decreased, the pH falsely increased, and the specimen will not liquefy. When part of the last portion of ejaculate is missing, the semen volume is decreased, the sperm count is falsely increased, the pH is falsely decreased, and the specimen will not clot. Patients should receive detailed instructions for specimen collection. Specimens are collected following a period of sexual abstinence of at least 2 days to not more than 7 days.1 Specimens collected following prolonged abstinence tend to have higher volumes and decreased motility.2 When performing fertility testing, the World Health Organization (WHO) recommends SUMMARY 10-1 Semen Production Structure Function Seminiferous tubules of testes Epididymis Ductus deferens Seminal vesicles Prostate gland Spermatogenesis Bulbourethral glands Sperm maturation Propel sperm to ejaculatory ducts Provide nutrients for sperm and fluid Provide enzymes and proteins for coagulation and liquefaction Add alkaline mucus to neutralize prostatic acid and vaginal acidity 205 that two or three samples be collected not less than 7 days or more than 3 weeks apart, with two abnormal samples considered significant. The laboratory should provide the patient with warm sterile glass or plastic containers. Whenever possible, the specimen is collected in a room provided by the laboratory. However, if this is not appropriate, the specimen should be kept at room temperature and delivered to the laboratory within 1 hour of collection. Laboratory personnel must record the patient’s name and birth date, the period of sexual abstinence, the completeness of the sample, difficulties with collection, and the times of specimen collection and specimen receipt. Specimens awaiting analysis should be kept at 37°C. Specimens should be collected by masturbation. If this is not possible, only nonlubricant-containing rubber or polyurethane condoms should be used. Ordinary condoms are not acceptable because they contain spermicides. TECHNICAL TIP Coitus interruptus is not a reliable means of semen collection because the first portion of the ejaculate, which contains the highest number of spermatozoa, may be lost and the low pH of the vaginal fluid may affect sperm motility.1 Specimen Handling All semen specimens are potential reservoirs for HIV and hepatitis viruses, and standard precautions must be observed at all times during analysis. Specimens are discarded as biohazardous waste. Sterile materials and techniques must be used when semen culture is to be performed or when the specimen is to be processed for bioassay, intra-uterine insemination (IUI), or in vitro fertilization (IVF).1 Semen Analysis Semen analysis for fertility evaluation consists of both macroscopic and microscopic examination. Parameters reported include appearance, volume, viscosity, pH, sperm concentration and count, motility, and morphology. Reference values are shown in Table 10–2. Appearance Normal semen has a gray-white color, appears translucent, and has a characteristic musty odor. When the sperm concentration is very low, the specimen may appear almost clear.1 Increased white turbidity indicates the presence of white blood cells (WBCs) and infection within the reproductive tract. If required, specimen culturing is performed prior to continuing with the semen analysis. During microscopic examination, WBCs must be differentiated from immature sperm (spermatids). The leukocyte esterase reagent strip test may be useful to screen for the presence of WBCs.3 Varying amounts of red coloration are associated with the presence of red blood cells (RBCs) and are abnormal. Yellow coloration may be caused by urine 3920_Ch10_203-216 23/01/14 10:31 AM Page 206 206 Part Three | Other Body Fluids Table 10–2 Reference Values for Semen Analysis5 Volume Viscosity pH Sperm concentration Sperm count Motility Quality Morphology Round cells 2 to 5 mL Pours in droplets 7.2 to 8.0 >20 million/mL >40 million/ejaculate >50% within 1 h >2.0 or a, b, c in Table 10–3 >14% normal forms (strict criteria) >30% normal forms (routine criteria) <1.0 million/mL contamination, specimen collection following prolonged abstinence, and medications. Urine is toxic to sperm, thereby affecting evaluation of motility. Liquefaction A fresh semen specimen is clotted and should liquefy within 30 to 60 minutes after collection; therefore, recording the time of collection is essential for evaluating semen liquefaction. Failure of liquefaction to occur within 60 minutes may be caused by a deficiency in prostatic enzymes and should be reported. Analysis of the specimen cannot begin until liquefaction has occurred. If after 2 hours the specimen has not liquified, an equal volume of physiologic Dulbecco’s phosphate-buffered saline (Procedure 10-1) or proteolytic enzymes such as alphachymotrypsin or bromelain (Procedure 10-2) may be added to induce liquefaction and allow the rest of the analysis to be performed. These treatments may affect biochemical tests, sperm motility, and sperm morphology, so their use must be documented. The dilution of semen with bromelain must be accounted for when calculating sperm concentration.1 Jelly-like granules (gelatinous bodies) may be present in liquefied semen specimens and have no clinical significance. Mucus strands, if present, may interfere with semen analysis.1 Volume Normal semen volume ranges between 2 and 5 mL. It can be measured by pouring the specimen into a clean graduated cylinder calibrated in 0.1-mL increments. Increased volume may be seen following periods of extended abstinence. Decreased volume is more frequently associated with infertility and may indicate improper functioning of one of the semenproducing organs, primarily the seminal vesicles. Incomplete specimen collection must also be considered. PROCEDURE 10-1 Semen Dilution With Physiologic Saline1 Prepare an equal volume of diluent and semen (1 part diluent and 1 part semen) using Dulbecco’s phosphate-buffered saline. Repeated pipetting of the prepared dilution will induce liquefaction. Preparation of Dulbecco’s Phosphate-Buffered Saline1 1. Using a 1-L volumetric flask, add the following: a. 750 ml of purified water b. 0.20 g of potassium chloride (KCL) c. 0.20 g of potassium dihydrogen phosphate (KH2PO4) d. 0.10 g of magnesium chloride hexahydrate (MgCl2.6H2O) e. 8.0 g of sodium chloride (NaCl) f. 2.16 g of disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O) g. 1.00 g of D-glucose 2. In a 10-mL volumetric flask, dissolve 0.132 g of calcium chloride dehydrate (CaCl2.2H2O) in 10 mL of purified water. 3. To prevent precipitation, add calcium chloride dehydrated solution to the 1-L flask slowly, stirring continuously. 4. Adjust the pH to 7.4 with 1 mol/L sodium hydroxide (NaOH). 5. Make up to 1000 mL with purified water. PROCEDURE 10-2 Digestion With Bromelain1 1. Prepare 10 IU/mL bromelain in Dulbecco’s phosphatebuffered saline. a. Into a 100-mL volumetric flask, add 1000 IU of bromelain. b. Add 60 mL Dulbecco’s phosphate-buffered saline. c. Mix to dissolve. It takes about 15 to 20 minutes. Bring volume to calibration mark using buffered saline. 2. Dilute one part semen 1:2 with the 10 IU/mL bromelain (1 part semen + 1 part bromelain solution). 3. Stir with a pipette tip. 4. Incubate at 37°C for 10 minutes. 5. Mix the sample well before analysis. Viscosity Specimen viscosity refers to the consistency of the fluid and may be related to specimen liquefaction. Incompletely liquefied specimens are clumped and highly viscous. The normal semen specimen should be easily drawn into a pipette and TECHNICAL TIP Gently mixing the sample container during liquefaction can help produce a homogeneous sample.1 3920_Ch10_203-216 23/01/14 10:31 AM Page 207 Chapter 10 | Semen form small discrete droplets that do not appear clumped or stringy when falling by gravity from the pipette. Droplets that form threads longer than 2 cm are considered highly viscous and are recorded as abnormal. Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity report.4 Viscosity can also be reported as low, normal, or high. Increased viscosity and incomplete liquefaction impede testing for sperm motility, sperm concentration, antisperm antibody detection, and measurement of biochemical markers.1 W W R Sperm Concentration and Sperm Count Even though fertilization is accomplished by one spermatozoon, the actual number of sperm present in a semen specimen is a valid measurement of fertility. Various factors can affect sperm concentration, such as the days of sexual abstinence before the collection, infection, or stress; therefore, more than one semen specimen should be evaluated for infertility studies. Reference values for sperm concentration are commonly listed as greater than 20 to 250 million sperm per milliliter; concentrations between 10 and 20 million per milliliter are considered borderline. The total sperm count for the ejaculate can be calculated by multiplying the sperm concentration by the specimen volume. Total sperm counts greater than 40 million per ejaculate are considered normal (20 million per milliliter × 2 mL). In the clinical laboratory, sperm concentration is usually performed using the Neubauer counting chamber. The sperm are counted in the same manner as cells in the cerebrospinal fluid cell count, that is, by diluting the specimen and counting the cells in the Neubauer chamber. The amount of the dilution and the number of squares counted vary among laboratories. The most commonly used dilution is 1:20 prepared using a mechanical (positive-displacement) pipette.5 Dilution of the semen is essential because it immobilizes the sperm before counting. The traditional diluting fluid contains sodium bicarbonate and formalin, which immobilize and preserve the cells; however, good results can also be achieved using saline and distilled water. Using the Neubauer hemocytometer, sperm are usually counted in the four corner and center squares of the large center square, similar to a manual RBC count (Fig. 10–2). Both sides of the hemocytometer are loaded and allowed to settle for 3 to 5 minutes; then they are counted, and the counts should agree within 10%. An average of the two counts is used R R pH The pH of semen indicates the balance between the pH values from the acidic prostatic secretion and the alkaline seminal vesicles secretion. The pH should be measured within 1 hour of ejaculation due to the loss of CO2 that occurs.1 The normal pH of semen is alkaline with a range of 7.2 to 8.0. Increased pH indicates infection within the reproductive tract. A decreased pH may be associated with increased prostatic fluid, ejaculatory duct obstruction, or poorly developed seminal vesicles.1 Semen for pH testing can be applied to the pH pad of a urinalysis reagent strip and the color compared with the manufacturer’s chart. Dedicated pH testing paper also can be used. 207 R W R W Figure 10–2 Areas of the Neubauer counting chamber used for red and white blood cell counts. W, typical WBC counting area; R, typical RBC counting area. in the calculation. If the counts do not agree, both the dilution and the counts are repeated. Counts are performed using either phase or bright-field microscopy. The addition of stain, such as crystal violet, to the diluting fluid aids in visualization when using bright-field microscopy. Only fully developed sperm should be counted. Immature sperm and WBCs, often referred to as “round” cells, must not be included. However, their presence can be significant, and they may need to be identified and counted separately. Stain included in the diluting fluid aids in differentiating between immature sperm cells (spermatids) and leukocytes, and they can be counted in the same manner as mature sperm. A count greater than 1 million leukocytes per milliliter is associated with inflammation or infection of the reproductive organs that can lead to infertility. The presence of more than 1 million spermatids per milliliter indicates disruption of spermatogenesis. This may be caused by viral infections, exposure to toxic chemicals, and genetic disorders. Calculating Sperm Concentration and Sperm Count Calculation of sperm concentration depends on the dilution used and the size and number of squares counted. When using the 1:20 dilution and counting the five squares (RBCs) in the large center square as described previously, the number of sperm can be multiplied by 1,000,000 (add 6 zeros) to equal the sperm concentration per milliliter. Notice that, unlike blood cell counts, the sperm concentration is reported in millions per milliliter rather than microliters. Sperm concentration can also be calculated using the basic formula for cell counts 3920_Ch10_203-216 23/01/14 10:31 AM Page 208 208 Part Three | Other Body Fluids covered in Chapter 9. Because this formula provides the number of cells per microliter, the figure must then be multiplied by 1000 to calculate the number of sperm per milliliter. The total sperm count is calculated by multiplying the number of sperm per milliliter by the specimen volume. EXAMPLES 1. Using a 1:20 dilution, an average of 60 sperm are counted in the five RBC counting squares on both sides of the hemocytometer. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 4 mL. 60 sperm counted × 1,000,000 = 60,000,000 sperm/mL 60,000,000 sperm/mL × 4 mL = 240,000,000 sperm/ ejaculate 2. In a 1:20 dilution, 600 sperm are counted in two WBC counting squares. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 2 µ L. 600 sperm counted × 20 (dilution) 60,000 sperm/µL = 2 sq mm × squares counted) × (volume counted) 0.1 mm (depth) 60,000 sperm/µL × 1000 = 60,000,000 sperm/mL 60,000,000/mL × 2 mL = 120,000,000 sperm/ejaculate Several methods have been developed using specially designed and disposable counting chambers that do not require specimen dilution. Comparison of these methods and the standard Neubauer counting chamber method showed poor correlation with the Neubauer method and also among themselves. The WHO states that the “validity of these alternative counting chambers must be established by checking chamber dimensions, comparing results with the improved Neubauer hemocytometer method, and obtaining satisfactory performance as shown by an external quality control program.”1 Sperm Motility The presence of sperm capable of forward, progressive movement is critical for fertility, because once presented to the cervix, the sperm must propel themselves through the cervical mucosa to the uterus, fallopian tubes, and ovum. Traditionally, clinical laboratory reporting of sperm motility has been a subjective evaluation performed by examining an undiluted specimen and determining the percentage of motile sperm and the quality of the motility. Sperm motility should be assessed using a well-mixed, liquefied semen specimen within 1 hour of specimen collection. The practice of examining sperm motility at timed intervals over an extended period has been shown to serve no useful purpose.6 To provide continuity in reporting, laboratories should place a consistent amount of semen on a slide under the same size cover slip, such as 10 µL under a 22 × 22 mm cover slip using a calibrated positive-displacement pipette, and allow it to settle for 1 minute. This procedure should be done in duplicate for accuracy. The percentage of sperm showing actual forward movement can then be estimated after evaluating approximately 20 high-power fields. An alternate procedure is to examine 200 sperm per slide and count the percentages of the different motile categories using a manual cell counter.1 Motility is evaluated by both speed and direction. Grading can be done using a scale of 0 to 4, with 4 indicating rapid, straight-line movement and 0 indicating no movement (Table 10–3). A minimum motility of 50% with a rating of 2.0 after 1 hour is considered normal.2 The WHO uses a rating scale of a, b, c, d (see Table 10–3). Interpretation states that within 1 hour, 50% or more of the sperm should be motile in categories a, b, and c, or 25% or more should show progressive motility (a and b).5 The WHO Laboratory Manual for the Examination and Processing of Human Semen (2010)1 currently recommends a simpler system for grading motility that does not include speed because of the difficulty in standardized reporting. Motility is graded as progressive motility (PM), nonprogressive motility (NP), and immotility (IM). Motility must be specified as total motility (PM and NP) or progressive motility (PM).1 (See Table 10–4.) The presence of a high percentage of immobile sperm and clumps of sperm requires further evaluation to determine sperm vitality or the presence of sperm agglutinins. In recent years, instrumentation capable of performing computer-assisted semen analysis (CASA) has been developed. Table 10–3 Grade Sperm Motility Grading WHO Criteria 4.0 3.0 a b 2.0 b 1.0 0 c d Table 10–4 Sperm Motility Action Rapid, straight-line motility Slower speed, some lateral movement Slow forward progression, noticeable lateral movement No forward progression No movement Alternative Sperm Motility Grading Criteria1 Progressive motility (PM) Nonprogressive motility (NP) Immotility (IM) Sperm moving linearly or in a large circle Sperm moving with an absence of progression No movement 3920_Ch10_203-216 23/01/14 10:31 AM Page 209 Chapter 10 | Semen CASA provides objective determination of both sperm velocity and trajectory (direction of motion). Sperm concentration and morphology are also included in the analysis. Currently, CASA instrumentation is found primarily in laboratories that specialize in andrology and perform a high volume of semen analysis. Sperm Morphology Just as the presence of a normal number of sperm that are nonmotile produces infertility, the presence of sperm that are morphologically incapable of fertilization also results in infertility. Sperm morphology is evaluated with respect to the structure of the head, neckpiece, midpiece, and tail. Abnormalities in head morphology are associated with poor ovum penetration, whereas neckpiece, midpiece, and tail abnormalities affect motility. The normal sperm has an oval-shaped head approximately 5 µm long and 3 µm wide and a long, flagellar tail approximately 45 µm long (Fig. 10–3). Critical to ovum penetration is the enzyme-containing acrosomal cap located at the tip of the head. The acrosomal cap should encompass approximately half of the head and cover approximately two thirds of the sperm nucleus.5 The neckpiece attaches the head to the tail and the midpiece. The midpiece is approximately 7.0 µm long and is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy required by the tail for motility. 209 Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion. Smears are made by placing approximately 10 µL of semen near the frosted end of a clean microscope slide. Place a second slide with a clean, smooth edge in front of the semen drop at a 45° angle and draw the slide back to the edge of the drop of semen, allowing the semen to spread across the end. When the semen is evenly distributed across the spreader slide, lightly pull the spreader slide forward with a continuous movement across the first slide to produce a smear. Staining can be performed using Wright’s, Giemsa, Shorr, or Papanicolaou stain and is a matter of laboratory preference. Air-dried slides are stable for 24 hours. At least 200 sperm should be evaluated and the percentage of abnormal sperm reported. Routinely identified abnormalities in head structure include double heads, giant and amorphous heads, pinheads, tapered heads, and constricted heads (Figs. 10–4 and 10–5). Abnormal sperm tails are frequently doubled, coiled, or bent (Figs. 10–6 and 10–7). An abnormally long neckpiece may cause the sperm head to bend backward and interfere with motility (Fig. 10–8). TECHNICAL TIP Sperm motility can be evaluated at room temperature or 37°C. When assessing motility at 37°C, the sample should be incubated at this temperature and the preparation made with prewarmed slides and cover slips.1 Acrosome Head Cell membrane Nucleus Figure 10–4 Spermatozoon with double head, hematoxylin-eosin (×1000). Midpiece Mitochondria Tail (flagellum) Figure 10–3 Normal spermatozoon structure. Figure 10–5 Spermatozoon with amorphous head, hematoxylin-eosin (×1000). 3920_Ch10_203-216 23/01/14 10:31 AM Page 210 210 Part Three | Other Body Fluids Calculating Round Cells Differentiation and enumeration of round cells (immature sperm and leukocytes) can also be made during the morphology examination (Fig. 10–9). Peroxidase-positive granulocytes are the predominant form of leukocyte in semen and can be further differentiated from spermatogenic cells and lymphocytes using a peroxidase stain. By counting the number of spermatids or leukocytes seen in conjunction with 100 mature sperm, the amount per milliliter can be calculated using the following formula, where N is the number of spermatids or neutrophils counted per 100 mature sperm, and S is the sperm concentration in millions per milliliter: Figure 10–6 Spermatozoon with double tail, hematoxylin-eosin (×1000). Additional parameters in evaluating sperm morphology include measuring head, neck, and tail size; measuring acrosome size; and evaluating for the presence of vacuoles. Inclusion of these parameters is referred to as Kruger’s strict criteria.7 Strict criteria evaluation requires the use of a stage micrometer or morphometry.8 At present, evaluation of sperm morphology using strict criteria is not routinely performed in the clinical laboratory but is recommended by the WHO.5 Strict criteria evaluation is an integral part of assisted reproduction evaluations. Normal values for sperm morphology depend on the evaluation method used and vary from greater than 30% normal forms when using routine criteria to greater than 14% normal forms when using strict criteria.5 Normal Double head Tapered head Constricted head Giant head Double tail Amorphous head Coiled tail C = N × S 100 This method can be used when counting cannot be performed during the hemocytometer count and to verify counts performed by hemocytometer. Greater than 1 million WBCs per milliliter per ejaculate indicates an inflammatory condition associated with infection and poor sperm quality and may impair sperm motility and DNA integrity.1 Additional Testing Should abnormalities be discovered in any of these routine parameters, additional tests may be requested (Table 10–5). The most common are tests for sperm vitality, seminal fluid fructose level, sperm agglutinins, and microbial infection. Pinhead Spermatid Figure 10–7 Common abnormalities of sperm heads and tails. 3920_Ch10_203-216 23/01/14 10:31 AM Page 211 Chapter 10 | Semen Table 10–5 211 Additional Testing for Abnormal Semen Analysis Abnormal Result Possible Abnormality Decreased motility with normal count Decreased count Vitality Eosin-nigrosin stain Fructose level Decreased motility with clumping Lack of seminal vesicle support medium Male antisperm antibodies Normal analysis with continued infertility Female antisperm antibodies Figure 10–8 Spermatozoon with bent neck and spermatid, hematoxylin-eosin (×1000). Test Mixed agglutination reaction and immunobead tests Sperm agglutination with male serum Sperm agglutination with female serum Figure 10–9 Immature spermatozoa, hematoxylin-eosin (×1000). Sperm Vitality Decreased sperm vitality may be suspected when a specimen has a normal sperm concentration with markedly decreased motility. Sperm vitality should be assessed within 1 hour of ejaculation. Vitality is evaluated by mixing the specimen with an eosin-nigrosin stain, preparing a smear, and counting the number of dead cells in 100 sperm using a brightfield or phase-contrast microscope. Living cells are not infiltrated by the dye and remain bluish white, whereas dead cells stain red against the purple background (Fig. 10–10). Normal vitality requires 50% or more living cells and should correspond to the previously evaluated motility. The presence of a large proportion of vital but immobile cells may indicate a defective flagellum, whereas a high number of immotile and nonviable cells may indicate epididymal pathology.1 Seminal Fluid Fructose Low sperm concentration may be caused by lack of the support medium produced in the seminal vesicles, which can be indicated by a low to absent fructose level in the Figure 10–10 Nonviable spermatozoa demonstrated by the eosinnigrosin stain (×1000). semen. Low fructose levels are caused by abnormalities of the seminal vesicles, bilateral congenital absence of the vas deferens, obstruction of the ejaculatory duct, partial retrograde ejaculation, and androgen deficiency. 1 Specimens can be screened for the presence of fructose using the resorcinol test that produces an orange color when fructose is present (Procedure 10-3). A normal quantitative level of fructose is equal to or greater than 13 µmol per ejaculate. This can be determined using spectrophotometric methods. Specimens for fructose levels should be tested within 2 hours of collection or frozen to prevent fructolysis. 3920_Ch10_203-216 23/01/14 10:32 AM Page 212 212 Part Three | Other Body Fluids PROCEDURE 10-3 Seminal Fructose Screening Test6 1. Prepare reagent (50 mg resorcinol in 33 mL concentrated HCl diluted to 100 mL with water). 2. Mix 1 mL of semen with 9 mL of reagent. 3. Boil. 4. Observe for orange-red color. Antisperm Antibodies Antisperm antibodies can be present in both men and women. They may be detected in semen, cervical mucosa, or serum, and are considered a possible cause of infertility. It is not unusual for both partners to demonstrate antibodies, although male antisperm antibodies are more frequently encountered. Under normal conditions, the blood–testes barrier separates sperm from the male immune system. When this barrier is disrupted, as can occur following surgery, vasectomy reversal (vasovasostomy), trauma, and infection, the antigens on the sperm produce an immune response that damages the sperm. The damaged sperm may cause the production of antibodies in the female partner.9 The presence of antibodies in a male subject can be suspected when clumps of sperm are observed during a routine semen analysis. Sperm-agglutinating antibodies cause sperm to stick to each other in a head-to-head, head-to-tail, or tailto-tail pattern.1 The agglutination is graded as “few,” “moderate,” or “many” on microscopic examination. The presence of antisperm antibodies in a female subject results in a normal semen analysis accompanied by continued infertility. The presence of antisperm antibodies in women may be demonstrated by mixing the semen with the female cervical mucosa or serum and observing for agglutination. A variety of immunoassay kits are available for both semen and serum testing. Two frequently used tests to detect the presence of antibody-coated sperm are the mixed agglutination reaction (MAR) test and the immunobead test. The MAR test is a screening procedure used primarily to detect the presence of immunoglobulin G (IgG) antibodies. The semen sample containing motile sperm is incubated with IgG antihuman globulin (AHG) and a suspension of latex particles or treated RBCs coated with IgG. The bivalent AHG binds simultaneously to both the antibody on the sperm and the antibody on the latex particles or RBCs, forming microscopically visible clumps of sperm and particles or cells. A finding of less than 10% of the motile sperm attached to the particles is considered normal. The immunobead test is a more specific procedure in that it can be used to detect the presence of IgG, IgM, and IgA antibodies and demonstrates what area of the sperm (head, neckpiece, midpiece, or tail) the autoantibodies are affecting. Head-directed antibodies can interfere with penetration into the cervical mucosa or ovum, whereas tail-directed antibodies affect movement through the cervical mucosa.10 In the immunobead test, sperm are mixed with polyacrylamide beads known to be coated with either anti-IgG, anti-IgM, or antiIgA. Microscopic examination of the sperm shows the beads attached to sperm at particular areas. Depending on the type of beads used, the test could be reported as “IgM tail antibodies,” “IgG head antibodies,” and so forth. The presence of beads on less than 50% of the sperm is considered normal as defined by the WHO.1 Microbial and Chemical Testing The presence of more than 1 million leukocytes per millimeter indicates infection within the reproductive system, frequently the prostate. Routine aerobic and anaerobic cultures and tests for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum are most frequently performed. Additional chemical testing performed on semen may include determining the levels of neutral α -glucosidase, free L-carnitine, glycerophosphocholine, zinc, citric acid, glutamyl transpeptidase, and prostatic acid phosphatase. Just as decreased fructose levels are associated with a lack of seminal fluid, decreased neutral α -glucosidase, glycerophosphocholine, and L-carnitine suggest a disorder of the epididymis. Decreased zinc, citric acid, glutamyl transpeptidase, and acid phosphatase indicate a lack of prostatic fluid (Table 10–6). Spectrophotometric methods are used to quantitate citric acid and zinc. On certain occasions, the laboratory may be called on to determine whether semen is actually present in a specimen. A primary example is in cases of alleged rape. Microscopically examining the specimen for the presence of sperm may be possible, with the best results being obtained by enhancing the specimen with xylene and examining under phase microscopy.11 Motile sperm can be detected for up to 24 hours after intercourse, whereas nonmotile sperm can persist for 3 days. As the sperm die off, only the heads remain and may be present for 7 days after intercourse. Seminal fluid contains a high concentration of prostatic acid phosphatase, so detecting this enzyme can aid in determining the presence of semen in a specimen. A more specific method is the detection of seminal glycoprotein p30 (prostatic specific antigen [PSA]), which is present even in the absence of sperm. 12 Further information can often be obtained by performing ABO blood grouping and DNA analysis on the specimen. Table 10–6 Reference Semen Chemical Values1 Neutral α -glucosidase Zinc Citric acid Acid phosphatase ≥ 20 mU/ejaculate ≥ 2.4 µmol/ejaculate ≥ 52 µmol/ejaculate ≥ 200 Units/ejaculate 3920_Ch10_203-216 23/01/14 10:32 AM Page 213 Chapter 10 | Semen Postvasectomy Semen Analysis Postvasectomy semen analysis is a much less involved procedure when compared with infertility analysis because the only concern is the presence or absence of spermatozoa. The length of time required for complete sterilization can vary greatly among patients and depends on both time and number of ejaculations. Therefore, finding viable sperm in a postvasectomy patient is not uncommon, and care should be taken not to overlook even a single sperm. Specimens are routinely tested at monthly intervals, beginning at 2 months postvasectomy and continuing until two consecutive monthly specimens show no spermatozoa. Recommended testing includes examining a wet preparation using phase microscopy for the presence of motile and nonmotile sperm. A negative wet preparation is followed by specimen centrifugation for 10 minutes and examination of the sediment.6 the motility and morphology analyses. The analysis is rated as a high complexity test under the Clinical Laboratory Improvement Amendments, and testing personnel standards must be observed. Increased interest in fertility testing has promoted the development of quality control materials and in-depth training programs. The standardized procedures developed by the WHO have provided a basis for laboratory testing and reporting. The use of CASA has aided in reducing the subjectivity of the analysis. However, even computerized analysis has been shown to vary among operators.15 Laboratories can now participate in proficiency testing programs offered by the College of American Pathologists and the American Association of Bioanalysts that include sperm concentration, vitality, and morphology. Commercial qualitycontrol materials and training aids are available and should be incorporated into laboratory protocols. Sperm Function Tests Advances in assisted reproduction and IVF have resulted in a need for more sophisticated semen analysis to assess not only the characteristics of sperm but also the functional ability. The tests are most commonly performed in specialized andrology laboratories and include the hamster egg penetration assay, cervical mucus penetration test, hypo-osmotic swelling test, and the in vitro acrosome reaction (Table 10–7).13 Semen Analysis Quality Control Traditionally, routine semen analysis has been subject to very little quality control,14 a situation that has resulted from a lack of appropriate control materials and the subjectivity of TECHNICAL TIP A single “motile” sperm on a wet preparation is an indication of an unsuccessful vasectomy. Table 10–7 Sperm Function Tests Test Description Hamster egg penetration Sperm are incubated with speciesnonspecific hamster eggs and penetration is observed microscopically Observation of sperm’s ability to penetrate partner’s midcycle cervical mucus Sperm exposed to low-sodium concentrations are evaluated for membrane integrity and sperm viability Evaluation of the acrosome to produce enzymes essential for ovum penetration Cervical mucus penetration Hypo-osmotic swelling In vitro acrosome reaction 213 Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. WHO Laboratory Manual for the Examination and Processing of Human Semen. World Health Organization. WHO Press, Geneva, Switzerland, 2010. 2. Sarhar, S, and Henry, JB: Andrology laboratory and fertility assessment. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 1996. 3. Lopez, A, et al: Suitability of solid-phase chemistry for quantification of leukocytes in cerebrospinal, seminal and peritoneal fluid. Clin Chem 33(8):1475–1476, 1987. 4. Overstreet, JW, and Katz, DF: Semen analysis. Urol Clin North Am 14(3):441–449, 1987. 5. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Interaction. Cambridge University Press, London, 1999. 6. Sampson, JH, and Alexander, NJ: Semen analysis: A laboratory approach. Lab Med 13(4):218–223, 1982. 7. Kruger, T, et al: Predictive value of sperm morphology in IVF. Fertil Steril 112–117, 1988. 8. Harr, R: Characterization of spermatozoa by planar morphometry. Clin Lab Sci 10(4):190–196, 1997. 9. Cearlock, DM: Autoimmune antispermatozoa antibodies in men: Clinical detection and role in infertility. Clin Lab Sci 2(3):165–168, 1989. 10. Marshburn, PB, and Kutteh, WH: The role of antisperm antibodies in infertility. Fertil Steril 61:799–811, 1994. 11. Fraysier, HD: A rapid screening technique for the detection of spermatozoa. J Forensic Sci 32(2):527–528, 1987. 12. Graves, HC, Sensabaugh, CF, and Blake, ET: Postcoital detection of a male-specific semen protein, application to the investigation of rape. N Engl J Med 312(6):338–343, 1985. 13. Yablonsky, T: Male fertility testing. Lab Med 27(6):378–383, 1996. 14. Baker, DJ, et al: Semen evaluations in the clinical laboratory. Lab Med 25(8):509–514, 1994. 15. Krause, W, and Viethen, G: Quality assessment of computer-assisted semen analysis (CASA) in the andrology laboratory. Andrologia 31(3):125–129, 1999. 3920_Ch10_203-216 23/01/14 10:32 AM Page 214 214 Part Three | Other Body Fluids Study Questions 1. Maturation of spermatozoa takes place in the: A. Sertoli cells B. Seminiferous tubules C. Epididymis D. Seminal vesicles 2. Enzymes for the coagulation and liquefaction of semen are produced by the: A. Seminal vesicles B. Bulbourethral glands C. Ductus deferens D. Prostate gland 3. The major component of seminal fluid is: A. Glucose B. Fructose C. Acid phosphatase D. Citric acid 4. If the first portion of a semen specimen is not collected, the semen analysis will have which of the following? A. Decreased pH B. Increased viscosity C. Decreased sperm count D. Decreased sperm motility 5. Failure of laboratory personnel to document the time a semen sample is collected primarily affects the interpretation of semen: A. Appearance B. Volume C. pH D. Viscosity 6. Liquefaction of a semen specimen should take place within: A. 1 hour B. 2 hours C. 3 hours D. 4 hours 7. A semen specimen delivered to the laboratory in a condom has a normal sperm count and markedly decreased sperm motility. This indicates: A. Decreased fructose B. Antispermicide in the condom C. Increased semen viscosity D. Increased semen alkalinity 8. An increased semen pH may be caused by: A. Prostatic infection B. Decreased prostatic secretions C. Decreased bulbourethral gland secretions D. All of the above 9. Proteolytic enzymes may be added to semen specimens to: A. Increase the viscosity B. Dilute the specimen C. Decrease the viscosity D. Neutralize the specimen 10. The normal sperm concentration is: A. Less than 20 million/µ L B. More than 20 million/mL C. Less than 20 million/mL D. More than 20 million/µ L 11. Given the following information, calculate the sperm concentration: dilution, 1:20; sperm counted in five RBC squares on each side of the hemocytometer, 80 and 86; volume, 3 mL. A. 80 million/mL B. 83 million/mL C. 86 million/mL D. 169 million/µ L 12. Using the above information, calculate the sperm concentration when 80 sperm are counted in 1 WBC square and 86 sperm are counted in another WBC square. A. 83 million/mL B. 166 million per ejaculate C. 16.6 million/mL D. 50 million per ejaculate 13. The primary reason to dilute a semen specimen before performing a sperm concentration is to: A. Immobilize the sperm B. Facilitate the chamber count C. Decrease the viscosity D. Stain the sperm 3920_Ch10_203-216 23/01/14 10:32 AM Page 215 Chapter 10 | Semen 14. When performing a sperm concentration, 60 sperm are counted in the RBC squares on one side of the hemocytometer and 90 sperm are counted in the RBC squares on the other side. The specimen is diluted 1:20. The: A. Specimen should be rediluted and counted B. Sperm count is 75 million/mL C. Sperm count is greater than 5 million/mL D. Sperm concentration is abnormal 15. Sperm motility evaluations are performed: A. Immediately after the specimen is collected B. Within 1 hour of collection C. After 3 hours of incubation D. At 6-hour intervals for 1 day 16. The percentage of sperm showing average motility that is considered normal is: A. 25% B. 50% C. 60% D. 75% 17. The purpose of the acrosomal cap is to: A. Penetrate the ovum B. Protect the the nucleus C. Create energy for tail movement D. Protect the neckpiece 18. The sperm part containing a mitochondrial sheath is the: A. Head B. Neckpiece C. Midpiece D. Tail 19. All of the following are associated with sperm motility except the: A. Head B. Neckpiece C. Midpiece D. Tail 20. The morphologic shape of a normal sperm head is: A. Round B. Tapered C. Oval D. Amorphous 21. Normal sperm morphology when using the WHO criteria is: A. >30% normal forms B. <30% normal forms C. >15% abnormal forms D. <15% normal forms 215 22. Additional parameters measured by Kruger’s strict morphology include all of the following except: A. Vitality B. Presence of vacuoles C. Acrosome size D. Tail length 23. Round cells that are of concern and may be included in sperm counts and morphology analysis are: A. Leukocytes B. Spermatids C. RBCs D. Both A and B 24. If 5 round cells per 100 sperm are counted in a sperm morphology smear and the sperm concentration is 30 million, the concentration of round cells is: A. 150,000 B. 1.5 million C. 300,000 D. 15 million 25. Following an abnormal sperm motility test with a normal sperm count, what additional test might be ordered? A. Fructose level B. Zinc level C. MAR test D. Eosin-nigrosin stain 26. Follow-up testing for a low sperm concentration would include testing for: A. Antisperm antibodies B. Seminal fluid fructose C. Sperm vitality D. Prostatic acid phosphatase 27. The immunobead test for antisperm antibodies: A. Detects the presence of male antibodies B. Determines the presence of IgG, IgM, and IgA antibodies C. Determines the location of antisperm antibodies D. All of the above 28. Measurement of α -glucosidase is performed to detect a disorder of the: A. Seminiferous tubules B. Epididymis C. Prostate gland D. Bulbourethral glands 3920_Ch10_203-216 23/01/14 10:32 AM Page 216 216 Part Three | Other Body Fluids 29. A specimen delivered to the laboratory with a request for prostatic acid phosphatase and glycoprotein p30 was collected to determine: A. Prostatic infection B. Presence of antisperm antibodies C. A possible rape D. Successful vasectomy 31. Standardization of procedures and reference values for semen analysis is primarily provided by the: A. Manufacturers of instrumentation B. WHO C. Manufacturers of control samples D. Clinical Laboratory Improvement Amendments 30. Following a negative postvasectomy wet preparation, the specimen should be: A. Centrifuged and reexamined B. Stained and reexamined C. Reported as no sperm seen D. Both A and B Case Studies and Clinical Situations 1. A repeat semen analysis for fertility testing is reported as follows: VOLUME: 3.5 mL SPERM COUNT: 6 million/mL VISCOSITY: Normal SPERM MOTILITY: 30%— grade 1.0 pH: 7.5 MORPHOLOGY: <30% normal forms—30 spermatids/100 sperm The results correspond with the first analysis. a. List three abnormal parameters. b. What is the sperm concentration? Is this normal? c. What is the spermatid count? Is this normal? d. Could the sperm concentration and the spermatid count be related to the infertility? Explain your answer. 2. A semen analysis on a vasovasostomy patient has a normal sperm concentration; however, motility is decreased, and clumping is observed on the wet preparation. a. Explain the possible connection between these observations and the patient’s recent surgery. b. What tests could be performed to further evaluate the patient’s infertility? c. Briefly explain the different interpretations offered by these two tests. d. State three ways in which a positive result on these tests could be affecting male fertility. 3. A yellow-colored semen specimen is received in the laboratory. The analysis is normal except for decreased sperm motility. Explain the possible connection between the two abnormal findings. 4. Abnormal results of a semen analysis are volume = 1.0 mL and sperm concentration = 1 million/mL. State a nonpathologic cause of these abnormal results. 5. A semen specimen with normal initial appearance fails to liquefy after 60 minutes. a. Would a specimen pH of 9.0 be consistent with this observation? Why or why not? b. State three chemical tests that would be of value in this analysis. c. How does this abnormality affect fertility? 6. A specimen is delivered to the laboratory with a request to determine if semen is present. a. What two chemical tests could be performed on the specimen? b. What additional examination could be performed on the specimen? 3920_Ch11_217-228 23/01/14 10:33 AM Page 217 CHAPTER 11 Synovial Fluid LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 11-1 Describe the formation and function of synovial fluid. 11-7 List and describe six crystals found in synovial fluid. 11-2 Relate laboratory test results to the four common classifications of joint disorders. 11-8 Explain the differentiation of monosodium urate and calcium pyrophosphate crystals using polarized and compensated polarized light. 11-3 State the five diagnostic tests most routinely performed on synovial fluid. 11-4 Determine the appropriate collection tubes for requested laboratory tests on synovial fluid. 11-5 Describe the appearance of synovial fluid in normal and abnormal states. 11-6 Discuss the normal and abnormal cellular composition of synovial fluid. 11-9 State the clinical significance of glucose and lactate tests on synovial fluid. 11-10 List four genera of bacteria most frequently found in synovial fluid. 11-11 Describe the relationship of serologic serum testing to joint disorders. KEY TERMS Arthritis Hyaluronic acid Synovial fluid Arthrocentesis Pseudogout Synoviocytes Gout 3920_Ch11_217-228 23/01/14 10:34 AM Page 218 218 Part Three | Other Body Fluids Physiology Table 11–1 Synovial fluid, often referred to as “joint fluid,” is a viscous liquid found in the cavities of the movable joints (diarthroses) or synovial joints. As shown in Figure 11–1, the bones in the synovial joints are lined with smooth articular cartilage and separated by a cavity containing the synovial fluid. The joint is enclosed in a fibrous joint capsule lined by the synovial membrane. The synovial membrane contains specialized cells called synoviocytes. The smooth articular cartilage and synovial fluid together reduce friction between the bones during joint movement. In addition to providing lubrication in the joints, synovial fluid provides nutrients to the articular cartilage and lessens the shock of joint compression that occurs during activities such as walking and jogging. Synovial fluid is formed as an ultrafiltrate of plasma across the synovial membrane. The filtration is nonselective except for the exclusion of high-molecular-weight proteins. Therefore, most of the chemical constituents, although seldom of clinical significance, have concentrations similar to plasma values. They do, however, provide nutrients for the vascular-deficient cartilage. The synoviocytes secrete a mucopolysaccharide containing hyaluronic acid and a small amount of protein (approximately one fourth of the plasma concentration) into the fluid. The large hyaluronate molecules contribute the noticeable viscosity to the synovial fluid. Damage to the articular membranes produces pain and stiffness in the joints, collectively referred to as arthritis. Laboratory results of synovial fluid analysis can be used to determine the pathologic origin of arthritis. The beneficial tests most frequently performed on synovial fluid are the white blood cell (WBC) count, differential, Gram stain, culture, and crystal examination.1 Reference values are shown in Table 11–1.2 A variety of conditions including infection, inflammation, metabolic disorders, trauma, physical stress, and advanced age are associated with arthritis. Disorders are frequently classified Normal Synovial Fluid Values2 Volume Color Clarity Viscosity Leukocyte count Neutrophils Crystals Glucose:plasma difference Total protein into four groups, as shown in Table 11–2. Some overlap of test results among the groups may occur (Table 11–3)3; the patient’s clinical history must also be considered when assigning a category. Specimen Collection and Handling Synovial fluid is collected by needle aspiration called arthrocentesis. The amount of fluid present varies with the size of the joint and the extent of fluid buildup in the joint. For example, the normal amount of fluid in the adult knee cavity is less than 3.5 mL, but can increase to greater than 25 mL with inflammation. In some instances, only a few drops of fluid are obtained, but these can still be used for microscopic analysis or culturing. The volume of fluid collected should be recorded. Table 11–2 Classification and Pathologic Significance of Joint Disorders Group Classification Pathologic Significance I. Noninflammatory Degenerative joint disorders, osteoarthritis II. Inflammatory Immunologic disorders, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, polymyositis, ankylosing spondylitis, rheumatic fever, Lyme arthritis Crystal-induced gout, pseudogout Microbial infection Traumatic injury, tumors, hemophilia, other coagulation disorders Anticoagulant overdose Synovial membrane Synovial cavity containing synovial fluid Articular cartilage Articular capsule Articulating bone Figure 11–1 A synovial joint. <3.5 mL Colorless to pale yellow Clear Able to form a string 4 to 6 cm long <200 cells/L <25% of the differential None present <10 mg/dL lower than the blood glucose level <3 g/dL III. Septic IV. Hemorrhagic 3920_Ch11_217-228 23/01/14 10:34 AM Page 219 Chapter 11 | Synovial Fluid Table 11–3 Laboratory Findings in Joint Disorders3 Table 11–4 219 Required Tube Types for Synovial Fluid Tests Group Classification Laboratory Findings Synovial Fluid Test Required Tube Type I. Noninflammatory Clear, yellow fluid Good viscosity WBCs <1000 L Neutrophils <30% Similar to blood glucose Gram stain and culture Cell counts Sterile heparinized or sodium polyanethol sulfonate Heparin or liquid ethylenediaminetetraacetic acid (EDTA) Sodium fluoride Nonanticoagulated II. Inflammatory Immunologic origin Crystal-induced origin III. Septic IV. Hemorrhagic Cloudy, yellow fluid Poor viscosity WBCs 2,000 to 75,000 L Neutrophils >50% Decreased glucose level Possible autoantibodies present Cloudy or milky fluid Low viscosity WBCs up to 100,000 L Neutrophils <70% Decreased glucose level Crystals present Cloudy, yellow-green fluid Variable viscosity WBCs 50,000 to 100,000 L Neutrophils >75% Decreased glucose level Positive culture and Gram stain Cloudy, red fluid Low viscosity WBCs equal to blood Neutrophils equal to blood Normal glucose level Glucose analysis All other tests TECHNICAL TIP Specimens for crystal analysis should not be refrigerated because they can produce additional crystals that can interfere with the identification of significant crystals. Color and Clarity A report of the gross appearance is an essential part of the synovial fluid analysis. Normal synovial fluid appears colorless to pale yellow. The word “synovial” comes from the Latin word for egg, ovum. Normal viscous synovial fluid resembles egg white. The color becomes a deeper yellow in the presence of noninflammatory and inflammatory effusions and may have a greenish tinge with bacterial infection. As with cerebrospinal fluid, in synovial fluid the presence of blood from a hemorrhagic arthritis must be distinguished from blood from a traumatic aspiration. This is accomplished primarily by observing the uneven distribution of blood or even a single blood streak in the specimens obtained from a traumatic aspiration. Turbidity is frequently associated with the presence of WBCs; however, synovial cell debris and fibrin also produce turbidity. The fluid may appear milky when crystals are present. Viscosity Normal synovial fluid does not clot; however, fluid from a diseased joint may contain fibrinogen and will clot. Therefore, fluid is often collected in a syringe that has been moistened with heparin. When sufficient fluid is collected, it should be distributed into specific tubes based on the required tests, as presented in Table 11–4. Powdered anticoagulants should not be used because they may produce artifacts that interfere with crystal analysis. The nonanticoagulated tube for other tests must be centrifuged and separated to prevent cellular elements from interfering with chemical and serologic analyses. Ideally, all testing should be done as soon as possible to prevent cellular lysis and possible changes in crystals. Synovial fluid viscosity comes from polymerization of the hyaluronic acid and is essential for the proper joints lubrication. Arthritis affects both the production of hyaluronate and its ability to polymerize, thus decreasing the fluid viscosity. Several methods are available to measure the synovial fluid viscosity, the simplest being to observe the fluid’s ability to form a string from the tip of a syringe, a test that easily can be done at the bedside. A string measuring 4 to 6 cm is considered normal. Hyaluronate polymerization can be measured using a Ropes, or mucin clot, test. When added to a solution of 2% to 5% acetic acid, normal synovial fluid forms a solid clot surrounded by clear fluid. As the ability of the hyaluronate to polymerize decreases, the clot becomes less firm, and the surrounding fluid increases in turbidity. The mucin clot test is 3920_Ch11_217-228 23/01/14 10:34 AM Page 220 220 Part Three | Other Body Fluids reported in terms of good (solid clot), fair (soft clot), low (friable clot), and poor (no clot). The mucin clot test is not routinely performed, because all forms of arthritis decrease viscosity and little diagnostic information is obtained. Formation of a mucin clot after adding acetic acid can be used to identify a questionable fluid as synovial fluid. Cell Counts The total leukocyte count is the most frequently performed cell count on synovial fluid. Red blood cell (RBC) counts are seldom requested. To prevent cellular disintegration, counts should be performed as soon as possible or the specimen should be refrigerated. Very viscous fluid may need to be pretreated by adding one drop of 0.05% hyaluronidase in phosphate buffer per milliliter of fluid and incubating at 37°C for 5 minutes. Manual counts on thoroughly mixed specimens are done using the Neubauer counting chamber. Clear fluids can usually be counted undiluted, but dilutions are necessary when fluids are turbid or bloody. Dilutions can be made using the procedure presented in Chapter 9; however, traditional WBC diluting fluid cannot be used because it contains acetic acid that causes the formation of mucin clots. Normal saline can be used as a diluent. If it is necessary to lyse the RBCs, hypotonic saline (0.3%) or saline that contains saponin is a suitable diluent. Methylene blue added to the normal saline stains the WBC nuclei, permitting separation of the RBCs and WBCs during counts performed on mixed specimens. The recommended technique is to line a petri dish with moist paper and place the hemocytometer on two small sticks to elevate it above the moist paper. Fill and count both sides of the hemocytometer for compatibility. Acceptable ranges are determined by the laboratory. Counting procedure: For counts less than 200 WBCs/L, count all 9 large squares. For counts greater than 200 WBCs /L in the above count, count the 4 corner squares. For counts greater than 200 WBCs /L in the above count, count the 5 small squares used for a RBC count.2 Table 11–5 Automated cell counters can be used for synovial fluid counts; however, highly viscous fluid may block the apertures, and the presence of debris and tissue cells may falsely elevate counts. As described previously, incubating the fluid with hyaluronidase decreases specimen viscosity. Analyzing scattergrams can aid in detecting tissue cells and debris. Properly controlled automated counts provide higher precision than manual counts.4 (See Appendix A.) WBC counts less than 200 cells/L are considered normal and may reach 100,000 cells/L or higher in severe infections.5 There is, however, considerable overlap of elevated leukocyte counts between septic and inflammatory forms of arthritis. Pathogenicity of the infecting organisms also produces varying results in septic arthritis, as does antibiotic administration. Differential Count Differential counts should be performed on cytocentrifuged preparations or on thinly smeared slides. Fluid should be incubated with hyaluronidase prior to slide preparation. Mononuclear cells, including monocytes, macrophages, and synovial tissue cells, are the primary cells seen in normal synovial fluid. Neutrophils should account for less than 25% of the differential count and lymphocytes less than 15%. Increased neutrophils indicate a septic condition, whereas an elevated cell count with a predominance of lymphocytes suggests a nonseptic inflammation. In both normal and abnormal specimens, cells may appear more vacuolated than they do on a blood smear.3 Besides increased numbers of these usually normal cells, other cell abnormalities include the presence of eosinophils, LE cells, Reiter cells (or neutrophages, vacuolated macrophages with ingested neutrophils), and RA cells (or ragocytes, neutrophils with small, dark cytoplasmic granules consisting precipitated rheumatoid factor). Lipid droplets may be present after crush injuries, and hemosiderin granules are seen in cases of pigmented villonodular synovitis. The most frequently encountered cells and inclusions seen in synovial fluid are summarized in Table 11–5. Cells and Inclusions Seen in Synovial Fluid Cell/Inclusion Description Significance Neutrophil Polymorphonuclear leukocyte Lymphocyte Macrophage (monocyte) Mononuclear leukocyte Large mononuclear leukocyte, may be vacuolated Synovial lining cell Similar to macrophage, but may be multinucleated, resembling a mesothelial cell LE cell Neutrophil containing characteristic ingested “round body” Vacuolated macrophage with ingested neutrophils Bacterial sepsis Crystal-induced inflammation Nonseptic inflammation Normal Viral infections Normal Disruption from arthrocentesis Lupus erythematosus Reiter cell Reactive arthritis (infection in another part of the body) 3920_Ch11_217-228 23/01/14 10:34 AM Page 221 Chapter 11 | Synovial Fluid Table 11–5 Cells and Inclusions Seen in Synovial Fluid—cont’d Cell/Inclusion Description Significance RA cell (ragocyte) Neutrophil with dark cytoplasmic granules containing immune complexes Cartilage cells Rice bodies Large, multinucleated cells Macroscopically resemble polished rice Microscopically show collagen and fibrin Refractile intracellular and extracellular globules Stain with Sudan dyes Inclusions within clusters of synovial cells Rheumatoid arthritis Immunologic inflammation Osteoarthritis Tuberculosis Septic and rheumatoid arthritis Traumatic injury Chronic inflammation Pigmented villonodular synovitis Fat droplets Hemosiderin Crystal Identification Microscopic examination of synovial fluid for the presence of crystals is an important diagnostic test in evaluating arthritis. Crystal formation in a joint frequently results in an acute, painful inflammation. It can also become a chronic condition. Causes of crystal formation include metabolic disorders and decreased renal excretion that produce elevated blood levels of crystallizing chemicals, degeneration of cartilage and bone, and injection of medications, such as corticosteroids, into a joint. Types of Crystals The primary crystals seen in synovial fluid are monosodium urate (uric acid) (MSU), found in cases of gout, and calcium pyrophosphate dihydrate (CPPD), seen with pseudogout. Increased serum uric acid resulting from impaired metabolism Table 11–6 221 of purines; increased consumption of high-purine-content foods, alcohol, and fructose; chemotherapy treatment of leukemias; and decreased renal excretion of uric acid are the most frequent causes of gout.5 Pseudogout is most often associated with degenerative arthritis, producing cartilage calcification and endocrine disorders that produce elevated serum calcium levels. Additional crystals that may be present include hydroxyapatite (basic calcium phosphate) associated with calcified cartilage degeneration, cholesterol crystals associated with chronic inflammation, corticosteroids after injections, and calcium oxalate crystals in renal dialysis patients. Patient history must always be considered. Characteristics and significance of the commonly encountered crystals are presented in Table 11–6. Artifacts present may include talcum powder and starch from gloves, precipitated anticoagulants, dust, and scratches on slides and cover slips. Slides and cover slips should be examined and if necessary cleaned again before use. Characteristics of Synovial Fluid Crystals Compensated Polarized Light Significance Needles Negative birefringence Gout Calcium pyrophosphate Rhomboid square, rods Positive birefringence Pseudogout Cholesterol Notched, rhomboid plates Negative birefringence Extracellular Crystal Shape Monosodium urate Image Continued 3920_Ch11_217-228 23/01/14 10:34 AM Page 222 222 Part Three | Other Body Fluids Table 11–6 Characteristics of Synovial Fluid Crystals—cont’d Image Compensated Polarized Light Crystal Shape Significance Corticosteroid Flat, variable-shaped plates Positive and negative birefringence Injections Calcium oxalate Envelopes Negative birefringence Renal dialysis Apatite (calcium phosphate) Small particles Require electron microscopy No birefringence Osteoarthritis Slide Preparation Ideally, crystal examination should be performed soon after fluid collection to ensure that crystals are not affected by changes in temperature and pH. Both MSU and CPPD crystals are reported as being located extracellularly and intracellularly (within neutrophils); therefore, fluid must be examined before WBC disintegration. Fluid is examined as an unstained wet preparation. One drop of fluid is placed on a precleaned glass slide and cover slipped. The slide can be initially examined under low and high power using a regular light microscope (Fig. 11–2). Crystals may be observed in Wright’s-stained smears (Fig. 11–3); however, this should not replace the wet prep examination and the use of polarized and red-compensated polarized light for identification. MSU crystals are routinely seen as needle-shaped crystals. They may be extracellular or located within the cytoplasm of neutrophils. They are frequently seen sticking through the cytoplasm of the cell. CPPD crystals usually appear rhomboid-shaped or square but may appear as short rods. They are usually located within vacuoles of the neutrophils, as shown in Figure 11–3. MSU crystals lyse phagosome membranes and therefore do not appear in vacuoles.6 To avoid misidentification of CPPD crystals, the classic rhomboid shape should be observed and confirmed with red compensated polarized microscopy. Figure 11–2 Unstained wet prep of MSU crystals (×400). Notice the characteristic yellow-brown of the urate crystals. Crystal Polarization Once the presence of the crystals has been determined using direct polarization, positive identification is made using firstorder red-compensated polarized light. A control slide for the polarization properties of MSU can be prepared using betamethasone acetate corticosteroid. Figure 11–3 Wright's-stained neutrophils containing CPPD crystals (×1000). 3920_Ch11_217-228 23/01/14 10:34 AM Page 223 Chapter 11 | Synovial Fluid 223 Both MSU and CPPD crystals have the ability to polarize light, as discussed in Chapter 6; however, MSU is more highly birefringent and appears brighter against the dark background (Figs. 11–4 and 11–5). When compensated polarized light is used, a red compensator is placed in the microscope between the crystal and the analyzer. The compensator separates the light ray into slowmoving and fast-moving vibrations and produces a red background (Fig. 11–6). Because of differences in the linear structure of the molecules in MSU and CPPD crystals, the color produced by each crystal when it is aligned with the slow vibration can be used to identify the crystal. The molecules in MSU crystals run parallel to the long axis of the crystal and, when aligned with the slow vibration, the velocity of the slow light passing through the crystal is not impeded as much as the fast light, which runs against the grain and produces a yellow color. This is considered negative birefringence (subtraction of velocity from the fast ray). In contrast, the molecules in CPPD crystals run perpendicular to the long axis of the crystal; when aligned with the slow axis of the compensator, the velocity of the fast light passing through the crystal is much quicker, producing a blue color and positive birefringence.7 When the crystals are aligned perpendicular to the slow vibration, the color is reversed, as shown in Figure 11–6. Care must be taken to ensure crystals being analyzed are aligned in accordance with the compensator axis. Notice how the colors of the MSU crystals in Figure 11–6 vary with the alignment. Figures 11–7 and 11–8 illustrate the characteristics of MSU and CPPD crystals under compensated polarized light. Figure 11–4 Strongly birefringent MSU crystals under polarized light (×500). Figure 11–7 MSU crystals under compensated polarized light. The yellow crystal is aligned with the slow vibration (×500). Figure 11–5 Weakly birefringent CPPD crystals under polarized light (×1000). Figure 11–8 CPPD crystals under compensated polarized light. The blue crystal is aligned with the slow vibration (×1000). Figure 11–6 Extracellular MSU crystals under compensated polarized light. Notice the change in color with crystal alignment (×100). 3920_Ch11_217-228 23/01/14 10:34 AM Page 224 224 Part Three | Other Body Fluids Crystal shapes and patterns of birefringence that vary from the standard MSU and CPPD patterns may indicate the presence of one of the less commonly encountered crystals and that further investigation is required (Fig. 11–9). Cholesterol, oxalate, and corticosteroid crystals exhibit birefringence, as do many contaminants. Apatite crystals are not birefringent.5 Chemistry Tests S ga low in ra st gr y ai n) (a (w Slow ith r gr ay ai n) Because synovial fluid is chemically an ultrafiltrate of plasma, chemistry test values are approximately the same as serum values. Therefore, few chemistry tests are considered clinically important. The most frequently requested test is the glucose determination, because markedly decreased glucose values indicate inflammatory (group II) or septic (group III) disorders. Because normal synovial fluid glucose values are based on the blood glucose level, simultaneous blood and synovial fluid samples should be obtained, preferably after the patient has fasted for 8 hours to allow equilibration between the two fluids. Under these conditions, normal synovial fluid glucose should not be more than 10 mg/dL lower than the blood value. A Other chemistry tests that may be requested are the total protein and uric acid determinations. Because the large protein molecules are not filtered through the synovial membranes, normal synovial fluid contains less than 3 g/dL protein (approximately one third of the serum value). Increased levels are found in inflammatory and hemorrhagic disorders; however, synovial fluid protein measurement does not contribute greatly to the classification of these disorders. When requested, the analysis is performed using the same methods used for serum protein determinations. The elevation of serum uric acid in cases of gout is well known; therefore, demonstration of an elevated synovial fluid uric acid level may be used to confirm the diagnosis when the presence of crystals cannot be demonstrated in the fluid. Serum uric acid is often measured as a first evaluation in suspected cases of gout. Fluid analysis for crystals is frequently still required. Fluid lactate or acid phosphatase levels maybe requested to monitor the severity and prognosis of rheumatoid arthritis (RA).2 Microbiologic Tests An infection may occur as a secondary complication of inflammation caused by trauma or through dissemination of a systemic infection; therefore, Gram stains and cultures are two of the most important tests performed on synovial fluid. Both tests must be performed on all specimens, as organisms are often missed on Gram stain. Bacterial infections are most frequently seen; however, fungal, tubercular, and viral infections also can occur. When they are suspected, special culturing procedures should be used. Patient history and other symptoms can aid in requests for additional testing. Routine bacterial cultures should include an enrichment medium, such as chocolate agar, because in addition to Staphylococcus and Streptococcus, the common organisms that infect synovial fluid are the fastidious Haemophilus species and Neisseria gonorrhoeae. S (w low ith ra gr y ain ) (ag Slo ain w ra st y gra in) Serologic Tests B Figure 11–9 Negative and positive birefringence in MSU and CPPD crystals. (A) MSU crystal with grain running parallel to the long axis. The slow ray passes with the grain, producing negative (yellow) birefringence. (B) CPPD crystal with grain running perpendicular to the long axis. The slow ray passes against the grain and is retarded, producing positive (blue) birefringence. TECHNICAL TIP To prevent falsely decreased values caused by glycolysis, specimens should be analyzed within 1 hour or preserved with sodium fluoride. Because of the association of the immune system to the inflammation process, serologic testing plays an important role in the diagnosis of joint disorders. However, most of these tests are performed on serum, and synovial fluid analysis actually serves as a confirmatory measure in cases that are difficult to diagnose. The autoimmune diseases rheumatoid arthritis and systemic lupus erythematosus cause very serious joint inflammation and are diagnosed in the serology laboratory by demonstrating the presence of their particular autoantibodies in the patient’s serum. These same antibodies can also be demonstrated in synovial fluid, if necessary. Arthritis is a frequent complication of Lyme disease. Therefore, demonstrating antibodies to the causative agent Borrelia burgdorferi in the patient’s serum can confirm the cause of the arthritis. Log on to www.fadavis.com/strasinger for additional content related to this chapter. 3920_Ch11_217-228 23/01/14 10:34 AM Page 225 Chapter 11 | Synovial Fluid References 1. Shmerling, RH: Synovial fluid analysis. A critical reappraisal. Rheum Dis Clin North Am 20(2):503–512, 1994. 2. Clinical and Laboratory Science Institute (CLSI): Analysis of Body Fluids in Clinical Chemistry, Approved Guideline, C49-A, Wayne, PA, 2007, CLSI. 3. Smith, GP, and Kjeldsberg, CR: Cerebrospinal, synovial, and serous body fluids. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 2001. 225 4. Brown, W, et al: Validation of body fluid analysis on the Coulter LH 750. Lab Hem 9(3):155–159, 2004. 5. Schumacher, HD, Clayburne, G, and Chen, L: Synovial fluid aspiration and analysis in evaluation of gout and other crystalinduced diseases. Arthritis Foundation: Bulletin on the Rheumatic Diseases 53(3), 2004. 6. Harris, MD, Siegel, LB, and Alloway, JA: Gout and hyperuricemia. Am Fam Physician 59(4):925–934, 1999. 7. Cornbleet, PJ: Synovial fluid crystal analysis. Lab Med 28(12): 774–779, 1997. Study Questions 1. The functions of synovial fluid include all of the following except: A. Lubrication for the joints B. Removal of cartilage debris C. Cushioning joints during jogging D. Providing nutrients for cartilage 2. The primary function of synoviocytes is to: A. Provide nutrients for the joints B. Secrete hyaluronic acid C. Regulate glucose filtration D. Prevent crystal formation 3. Which of the following is not a frequently performed test on synovial fluid? A. Uric acid B. WBC count C. Crystal examination D. Gram stain 4. The procedure for collecting synovial fluid is called: A. Synovialcentesis B. Arthrocentesis C. Joint puncture D. Arteriocentesis 5. Match the following disorders with their appropriate group: A. Noninflammatory B. Inflammatory C. Septic D. Hemorrhagic ____ Gout ____ Neisseria gonorrhoeae infection ____ Systemic lupus erythematosus ____ Osteoarthritis ____ Hemophilia ____ Rheumatoid arthritis ____ Heparin overdose 6. Normal synovial fluid resembles: A. Egg white B. Normal serum C. Dilute urine D. Lipemic serum 7. Before testing, very viscous synovial fluid should be treated with: A. B. C. D. Normal saline Hyaluronidase Distilled water Hypotonic saline 8. Addition of a cloudy, yellow synovial fluid to acetic acid produces a/an: A. Yellow-white precipitate B. Easily dispersed clot C. Solid clot D. Opalescent appearance 9. Which of the following could be the most significantly affected if a synovial fluid is refrigerated before testing? A. Glucose B. Crystal examination C. Mucin clot test D. Differential 10. The highest WBC count can be expected to be seen with: A. B. C. D. Noninflammatory arthritis Inflammatory arthritis Septic arthritis Hemorrhagic arthritis 11. When diluting a synovial fluid WBC count, all of the following are acceptable except: A. Acetic acid B. Isotonic saline C. Hypotonic saline D. Saline with saponin 3920_Ch11_217-228 23/01/14 10:34 AM Page 226 226 Part Three | Other Body Fluids 12. The lowest percentage of neutrophils would be seen in: A. Noninflammatory arthritis B. Inflammatory arthritis C. Septic arthritis D. Hemorrhagic arthritis 13. All of the following are abnormal when seen in synovial fluid except: A. Neutrophages B. Ragocytes C. Synovial lining cells D. Lipid droplets 14. Synovial fluid crystals that occur as a result of purine metabolism or chemotherapy for leukemia are: A. Monosodium urate B. Cholesterol C. Calcium pyrophosphate D. Apatite 15. Synovial fluid crystals associated with inflammation in dialysis patients are: A. Calcium pyrophosphate dihydrate B. Calcium oxalate C. Corticosteroid D. Monosodium urate 16. Crystals associated with pseudogout are: A. Monosodium urate B. Calcium pyrophosphate dihydrate C. Apatite D. Corticosteroid 17. Synovial fluid for crystal examination should be examined as a/an: A. Wet preparation B. Wright's stain C. Gram stain D. Acid-fast stain 18. Crystals that have the ability to polarize light are: A. Corticosteroid B. Monosodium urate C. Calcium oxalate D. All of the above 19. In an examination of synovial fluid under compensated polarized light, rhomboid-shaped crystals are observed. What color would these crystals be when aligned parallel to the slow vibration? A. White B. Yellow C. Blue D. Red 20. If crystals shaped like needles are aligned perpendicular to the slow vibration of compensated polarized light, what color are they? A. White B. Yellow C. Blue D. Red 21. Negative birefringence occurs under red-compensated polarized light when: A. Slow light is impeded more than fast light B. Slow light is less impeded than fast light C. Fast light runs against the molecular grain of the crystal D. Both B and C 22. Synovial fluid cultures are often plated on chocolate agar to detect the presence of: A. Neisseria gonorrhoeae B. Staphylococcus agalactiae C. Streptococcus viridans D. Enterococcus faecalis 23. The most frequently performed chemical test on synovial fluid is: A. Total protein B. Uric acid C. Calcium D. Glucose 24. Which of the following chemistry tests can be performed on synovial fluid to determine the severity of RA? A. Glucose B. Protein C. Lactate D. Uric acid 25. Serologic tests on patients’ serum may be performed to detect antibodies causing arthritis for all of the following disorders except: A. Pseudogout B. Rheumatoid arthritis C. Systemic lupus erythematosus D. Lyme arthritis 3920_Ch11_217-228 23/01/14 10:34 AM Page 227 Chapter 11 | Synovial Fluid 227 Case Studies and Clinical Situations 1. A 50-year-old man presents in the emergency department with severe pain and swelling in the right knee. Arthrocentesis is performed and 20 mL of milky synovial fluid is collected. The physician orders a Gram stain, culture, and crystal examination of the fluid, as well as a serum uric acid. She requests that the synovial fluid be saved for possible additional tests. a. Describe the tubes into which the fluid would be routinely placed. b. If the patient’s serum uric acid level is elevated, what type of crystals and disorder are probable? c. Describe the appearance of these crystals under direct and compensated polarized light. d. Why were the Gram stain and culture ordered? 2. A medical laboratory science student dilutes a synovial fluid sample before performing a WBC count. The fluid forms a clot. a. Why did the clot form? b. How can the student perform a correct dilution of the fluid? c. After the correct dilution is made, the WBC count is 100,000/L. State two arthritis classifications that could be considered. d. State two additional tests that could be run to determine the classification. 3. Fluid obtained from the knee of an obese 65-year-old woman being evaluated for a possible knee replacement has the following results: APPEARANCE: Pale yellow and hazy WBC COUNT: 500 cells/L GRAM STAIN: Negative GLUCOSE: 110 mg/dL (serum glucose: 115 mg/dL) a. What classification of joint disorder do these results suggest? b. Under electron microscopy, what crystals might be detected? c. How does the glucose result aid in the disorder classification? 4. A synovial fluid sample delivered to the laboratory for a cell count is clotted. a. What abnormal constituent is present in the fluid? b. What type of tube should be sent to the laboratory for a cell count? c. Could the original tube be used for a Gram stain and culture? Why or why not? 3920_Ch11_217-228 23/01/14 10:34 AM Page 228 3920_Ch12_229-242 23/01/14 10:35 AM Page 229 CHAPTER 12 Serous Fluid LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 12-1 Describe the normal formation of serous fluid. 12-2 Describe four primary causes of serous effusions. 12-3 Differentiate between a transudate and an exudate, including etiology, appearance, and laboratory tests. 12-4 Differentiate between a hemothorax and a hemorrhagic exudate. 12-5 Differentiate between a chylous and a pseudochylous exudate. 12-6 State the significance of increased neutrophils, lymphocytes, eosinophils, and plasma cells in pleural fluid. 12-7 Describe the morphologic characteristics of mesothelial cells and malignant cells. 12-8 List three common chemistry tests performed on pleural fluid, and state their significance. 12-9 State the common etiologies of pericardial effusions. 12-10 Discuss the diagnostic significance of peritoneal lavage. 12-11 Calculate a serum-ascites gradient, and state its significance. 12-12 Differentiate between ascitic effusions of hepatic and peritoneal origin. 12-13 State the clinical significance of the carcinoembryonic antigen and CA 125 tests. 12-14 List four chemical tests performed on ascitic fluid, and state their significance. KEY TERMS Ascites Paracentesis Ascitic fluid Parietal membrane Serum-ascites albumin gradient (SAAG) Effusion Pericardiocentesis Thoracentesis Exudate Pericarditis Transudate Hydrostatic pressure Peritonitis Visceral membrane Oncotic pressure Serous fluid 3920_Ch12_229-242 23/01/14 10:35 AM Page 230 230 Part Three | Other Body Fluids The closed cavities of the body—namely, the pleural, pericardial, and peritoneal cavities—are each lined by two membranes referred to as the serous membranes. One membrane lines the cavity wall (parietal membrane), and the other covers the organs within the cavity (visceral membrane) (Fig. 12–1). The fluid between the membranes is called serous fluid, and it provides lubrication between the parietal and visceral membranes. Lubrication is necessary to prevent the friction between the two membranes that occurs as a result of movement of the enclosed organs, such as in the expansion and contraction of the lungs. Normally, only a small amount of serous fluid is present, because production and reabsorption take place at a constant rate. pressure in the parietal and visceral capillaries creates a small excess of fluid that is reabsorbed by the lymphatic capillaries located in the membranes. In Figure 12–2, the normal formation and absorption of pleural fluid are demonstrated. Disruption of the mechanisms of serous fluid formation and reabsorption causes an increase in fluid between the membranes. This increase is termed an effusion. Primary causes of effusions include increased hydrostatic pressure (congestive heart failure), decreased oncotic pressure (hypoproteinemia), increased capillary permeability (inflammation and infection), and lymphatic obstruction (tumors) (Table 12–1). Specimen Collection and Handling Formation Serous fluids are formed as ultrafiltrates of plasma; no additional material is contributed by the mesothelial cells that line the membranes. Production and reabsorption are subject to hydrostatic pressure and colloidal pressure (oncotic pressure) from the capillaries that serve the cavities and the capillary permeability. Under normal conditions, colloidal pressure from serum proteins is the same in the capillaries on both sides of the membrane. Therefore, hydrostatic pressure in the parietal and visceral capillaries causes fluid to enter between the membranes. Filtration of the plasma ultrafiltrate results in increased oncotic pressure in the capillaries that favors reabsorption of fluid back into the capillaries. This action produces a continuous exchange of serous fluid and maintains the normal volume of fluid between the membranes. The slightly different amount of positive Parietal pericardium Pericardial cavity Visceral pericardium Fluids for laboratory examination are collected by needle aspiration from the respective cavities. These aspiration procedures are referred to as thoracentesis (pleural), pericardiocentesis (pericardial), and paracentesis (peritoneal). Abundant fluid (>100 mL) is usually collected; therefore, suitable specimens are available for each section of the laboratory. An EDTA tube is used for cell counts and the differential. Sterile heparinized or sodium polyanethol sulfonate (SPS) evacuated tubes are used for microbiology and cytology. For better recovery of microorganisms and abnormal cells, concentration of large amounts of fluid is performed by centrifugation. Chemistry tests can be run on clotted specimens in plain tubes or in heparin tubes. Specimens for pH must be maintained anaerobically in ice.1 Chemical tests performed on Parietal pleura Pleural cavity Visceral pleura Lung Body wall Liver Stomach Parietal peritoneum Visceral peritoneum Intestine Peritoneal cavity Figure 12–1 The body areas and membranes where serous fluid is produced. 3920_Ch12_229-242 23/01/14 10:35 AM Page 231 Chapter 12 | Serous Fluid serous fluids are frequently compared with plasma chemical concentrations because the fluids are essentially plasma ultrafiltrates. Therefore, blood specimens should be obtained at the time of collection. Parietal pleura Chest wall Visceral pleura Pleural space Lung Systemic capillary Pulmonary capillary Oncotic pressure Oncotic pressure (mm Hg) – 26 26 + Hydrostatic pressure (mm Hg) +30 Intrapleural pressure Hydrostatic pressure +5 +9 –5 +10 –11 Intrapleural pressure Lymphatic system 231 Transudates and Exudates A general classification of the cause of an effusion can be accomplished by categorizing the fluid as either transudate or exudate. Effusions that form because of a systemic disorder that disrupts the balance in the regulation of fluid filtration and reabsorption—such as the changes in hydrostatic pressure created by congestive heart failure or the hypoproteinemia associated with the nephrotic syndrome—are called transudates. Exudates are produced by conditions that directly involve the membranes of the particular cavity, including infections and malignancies. Classifying a serous fluid as a transudate or exudate can provide a valuable initial diagnostic step and aid in the course of further laboratory testing, because it is usually not necessary to test transudate fluids.2 Traditionally, a variety of laboratory tests have been used to differentiate between transudates and exudates, including appearance, total protein, lactic dehydrogenase, cell counts, and spontaneous clotting. However, the most reliable differentiation is usually obtained by determining the fluid: blood ratios for protein and lactic dehydrogenase.3 Differential values for these parameters are shown in Table 12–2. Additional tests are available for specific fluids and will be discussed in the following sections. General Laboratory Procedures Serous fluid examination—including classification as a transudate or exudate; appearance; cell count and differential; and chemistry, microbiology, and cytology procedures—is Figure 12–2 The normal formation and absorption of pleural fluid. Table 12–2 Table 12–1 Laboratory Differentiation of Transudates and Exudates Pathologic Causes of Effusions Increased capillary hydrostatic pressure Congestive heart failure Salt and fluid retention Decreased oncotic pressure Nephrotic syndrome Hepatic cirrhosis Malnutrition Protein-losing enteropathy Increased capillary permeability Microbial infections Membrane inflammations Malignancy Lymphatic obstruction Malignant tumors, lymphomas Infection and inflammation Thoracic duct injury Appearance Fluid:serum protein ratio Fluid:serum LD ratio WBC count Spontaneous clotting Pleural fluid cholesterol Pleural fluid: serum cholesterol ratio Pleural fluid: bilirubin ratio Serum-ascites albumin gradient Transudate Exudate Clear <0.5 Cloudy >0.5 <0.6 >0.6 <1000/µL No >1000/µL Possible <45 to 60 mg/dL <0.3 >45 to 60 mg/dL >0.3 <0.6 >0.6 >1.1 <1.1 3920_Ch12_229-242 23/01/14 10:35 AM Page 232 232 Part Three | Other Body Fluids performed in the same manner on all serous fluids. However, the significance of the test results and the need for specialized tests vary among fluids. Therefore, the interpretation of routine and special procedures will be discussed individually for each of the three serous fluids. Tests that are usually performed on all serous fluids include evaluation of the appearance and differentiation between a transudate and an exudate. Effusions of exudative origin are then examined for the presence of microbiologic and cytologic abnormalities. Additional tests are ordered based on specific clinical symptoms. Red blood cell (RBC) and white blood cell (WBC) counts are not routinely performed on serous fluids because they provide little diagnostic information.4 In general, WBC counts greater than 1000/L and RBC counts greater than 100,000/L indicate an exudate. Serous fluid cell counts can be performed manually by using a Neubauer counting chamber and the methods discussed in Chapter 9 or by electronic cell counters (see Appendix A). When manual cell counts are performed, they frequently include a count of all nucleated cells.1 Inclusion of tissue cells and debris in the count must be considered when electronic counters are used, and care must be taken to prevent the blocking of tubing with debris. Differential cell counts are routinely performed on serous fluids, preferably on Wright’s-stained, cytocentrifuged specimens or on slides prepared from the sediment of centrifuged specimens. Smears must be examined not only for WBCs, but also for normal and malignant tissue cells. Any suspicious cells seen on the differential are referred to the cytology laboratory or the pathologist. Pleural Fluid Pleural fluid is obtained from the pleural cavity, located between the parietal pleural membrane lining the chest wall and the visceral pleural membrane covering the lungs. Pleural effusions may be either transudative or exudative. In addition to the tests routinely performed to differentiate between transudates and exudates, two additional procedures are helpful when analyzing pleural fluid: the pleural fluid cholesterol and fluid:serum cholesterol ratio and the pleural fluid:serum total bilirubin ratio. A pleural fluid cholesterol >60 mg/dL or a pleural fluid:serum cholesterol ratio >0.3 provides reliable information that the fluid is an exudate.5 A fluid:serum total bilirubin ratio of 0.6 or more also indicates the presence of an exudate. Appearance Considerable diagnostic information concerning the etiology of a pleural effusion can be learned from the specimen appearance (Table 12–3). Normal and transudate pleural fluids are clear and pale yellow. Turbidity is usually related to the presence of WBCs and indicates bacterial infection, tuberculosis, or an immunologic disorder such as rheumatoid arthritis. The presence of blood in the pleural fluid can signify a hemothorax (traumatic injury), membrane damage such as occurs in malignancy, or a traumatic aspiration. As seen with Table 12–3 Appearance Correlation of Pleural Fluid Appearance and Disease5 Disorder Clear, pale yellow Normal Turbid, white Microbial infection (tuberculosis) Bloody Hemothorax Hemorrhagic effusion, pulmonary embolus, tuberculosis, malignancy Milky Chylous material from thoracic duct leakage Pseudochylous material from chronic inflammation Brown Rupture of amoebic liver abscess Black Aspergillus Viscous Malignant mesothelioma (increased hyaluronic acid) other fluids, blood from a traumatic tap appears streaked and uneven. To differentiate between a hemothorax and hemorrhagic exudate, a hematocrit can be run on the fluid. If the blood is from a hemothorax, the fluid hematocrit is more than 50% of the whole blood hematocrit, because the effusion comes from the inpouring of blood from the injury.6 A chronic membrane disease effusion contains both blood and increased pleural fluid, resulting in a much lower hematocrit. The appearance of a milky pleural fluid may be due to the presence of chylous material from thoracic duct leakage or to pseudochylous material produced in chronic inflammatory conditions. Chylous material contains a high concentration of triglycerides, whereas pseudochylous material has a higher concentration of cholesterol. Therefore, Sudan III staining is strongly positive with chylous material. In contrast, pseudochylous effusions contain cholesterol crystals.5 Differentiation between chylous and pseudochylous effusions is summarized in Table 12–4. Hematology Tests As mentioned previously, the differential cell count is the most diagnostically significant hematology test performed on serous fluids. Primary cells associated with pleural fluid include macrophages, neutrophils, lymphocytes, eosinophils, mesothelial cells, plasma cells, and malignant cells. Macrophages normally account for 64% to 80% of a nucleated cell count followed by lymphocytes (18% to 30%) and neutrophils (1% to 2%) (Table 12–5). These same cells are also found in pericardial and peritoneal fluids. Similar to other body fluids, an increase in pleural fluid neutrophils indicates a bacterial infection, such as pneumonia. Neutrophils are also increased in effusions resulting from pancreatitis and pulmonary infarction. 3920_Ch12_229-242 23/01/14 10:35 AM Page 233 Chapter 12 | Serous Fluid Table 12–4 Differentiation Between Chylous and Pseudochylous Pleural Effusions Chylous Effusion Cause Thoracic duct damage Appearance Leukocytes Milky/white Predominantly lymphocytes Absent Cholesterol crystals Triglycerides Sudan III staining Table 12–5 233 >110 mg/dL Strongly positive Pseudochylous Effusion Chronic inflammation Lymphatic obstruction Milky/green tinge Mixed cells Present <50 mg/dL Negative/weakly positive Significance of Cells Seen in Pleural Fluid Cells Significance Neutrophils Pancreatitis Pulmonary infarction Tuberculosis Viral infection Autoimmune disorders Malignancy Normal and reactive forms have no clinical significance Decreased mesothelial cells are associated with tuberculosis Tuberculosis Primary adenocarcinoma and small-cell carcinoma Metastatic carcinoma Lymphocytes Mesothelial cells Plasma cells Malignant cells Lymphocytes are normally noticeably present in both transudates and exudates in a variety of forms, including small, large, and reactive. More prominent nucleoli and cleaved nuclei may be present. Elevated lymphocyte counts are seen in effusions resulting from tuberculosis, viral infections, malignancy, and autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus. Systemic lupus erythematosus cells may be seen (Fig. 12–3). Increased eosinophil levels (>10%) may be associated with trauma resulting in the presence of air or blood (pneumothorax and hemothorax) in the pleural cavity. They are also seen in allergic reactions and parasitic infections. Figure 12–3 Systemic lupus erythematosus cell in pleural fluid. Notice the ingested “round body” (×1000). The membranes lining the serous cavities contain a single layer of mesothelial cells, so it is not unusual to find these cells in the serous fluids. Mesothelial cells are pleomorphic; they resemble lymphocytes, plasma cells, and malignant cells, frequently making identification difficult. They often appear as single small or large round cells with abundant blue cytoplasm and round nuclei with uniform dark purple cytoplasm and may be referred to as “normal” mesothelial cells (Figs. 12–4 and 12–5). In contrast, “reactive” mesothelial cells may appear in clusters; have varying amounts of cytoplasm, eccentric nuclei, and prominent nucleoli; and be multinucleated, thus more closely resembling malignant cells (Figs. 12–6 and 12–7). An increase in mesothelial cells is not a diagnostically significant finding, but they may be increased in pneumonia and malignancy. Of more significance is the noticeable lack of mesothelial cells associated with tuberculosis, which results from exudate covering the pleural membranes. Also associated with tuberculosis is an increase in the presence of pleural fluid plasma cells (Fig. 12–8). A primary concern in examining all serous effusions is detecting malignant cells. Differentiating among mesothelial cells and other tissue cells and malignant cells is often Figure 12–4 Normal pleural fluid mesothelial cells, lymphocytes, and monocytes (×250). 3920_Ch12_229-242 23/01/14 10:35 AM Page 234 234 Part Three | Other Body Fluids Figure 12–5 Normal mesothelial cell (×500). Figure 12–6 Reactive mesothelial cells showing eccentric nuclei and vacuolated cytoplasm (×500). Figure 12–7 One normal and two reactive mesothelial cells with a multinucleated form (×500). difficult. Distinguishing characteristics of malignant cells may include nuclear and cytoplasmic irregularities, hyperchromatic nucleoli, cellular clumps with cytoplasmic molding (community borders), and abnormal nucleus:cytoplasm ratios (Figs. 12–9 to 12–11). Malignant pleural effusions most frequently contain large, irregular adenocarcinoma Figure 12–8 Pleural fluid plasma cells seen in a case of tuberculosis. Notice the absence of mesothelial cells (×1000). Figure 12–9 Pleural fluid adenocarcinoma showing cytoplasmic molding (×250). Figure 12–10 Pleural fluid adenocarcinoma showing nuclear and cytoplasmic molding and vacuolated cytoplasm (×1000). cells, small or oat cell carcinoma cells resembling large lymphocytes, and clumps of metastatic breast carcinoma cells (Figs. 12–12 to 12–14). Special staining techniques and flow cytometry may be used for positive identification of tumor cells. Table 12–6 describes the primary characteristics of malignant serous fluid cells. 3920_Ch12_229-242 23/01/14 10:35 AM Page 235 Chapter 12 | Serous Fluid Figure 12–11 Enhancement of nuclear irregularities using a toluidine blue stain (×250). Figure 12–14 Metastatic breast carcinoma cells in pleural fluid. Notice the hyperchromatic nucleoli (×1000). Table 12–6 Figure 12–12 Poorly differentiated pleural fluid adenocarcinoma showing nuclear irregularities and cytoplasmic vacuoles (×500). Chemistry Tests In addition to the chemical tests performed to differentiate between a pleural transudate and a pleural exudate, the most common chemical tests performed on pleural fluid are glucose, pH, adenosine deaminase (ADA), and amylase (Table 12–7). Characteristics of Malignant Cells Increased nucleus:cytoplasm (N:C) ratio. The higher the ratio, the more poorly differentiated are the cells. Irregularly distributed nuclear chromatin Variation in size and shape of nuclei Increased number and size of nucleoli Hyperchromatic nucleoli Giant cells and multinucleation Nuclear molding Cytoplasmic molding (community borders) Vacuolated cytoplasm, mucin production Cellular crowding, phagocytosis Table 12–7 Figure 12–13 Pleural fluid small cell carcinoma showing nuclear molding (×250). 235 Significance of Chemical Testing of Pleural Fluid Test Significance Glucose Decreased in rheumatoid inflammation Decreased in purulent infection Elevated in bacterial infection Elevated in chylous effusions Decreased in pneumonia not responding to antibiotics Markedly decreased with esophageal rupture Elevated in tuberculosis and malignancy Elevated in pancreatitis, esophageal rupture, and malignancy Lactate Triglyceride pH ADA Amylase 3920_Ch12_229-242 23/01/14 10:35 AM Page 236 236 Part Three | Other Body Fluids Triglyceride levels may also be measured to confirm the presence of a chylous effusion. Decreased glucose levels are seen with tuberculosis, rheumatoid inflammation, and purulent infections. As an ultrafiltrate of plasma, pleural fluid glucose levels parallel plasma levels, and values less than 60 mg/dL are considered decreased. Fluid values should be compared with plasma values. Pleural fluid lactate levels are elevated in bacterial infections and can be considered in addition to the glucose level. Pleural fluid pH lower than 7.2 may indicate the need for chest-tube drainage, in addition to administration of antibiotics in cases of pneumonia. In cases of acidosis, the pleural fluid pH should be compared with the blood pH. Pleural fluid pH at least 0.30 degrees lower than the blood pH is considered significant.7 A pH value as low as 6.0 indicates an esophageal rupture that is allowing the influx of gastric fluid. ADA levels higher than 40 U/L are highly indicative of tuberculosis. They are also frequently elevated with malignancy. As with serum, elevated amylase levels are associated with pancreatitis, and amylase is often elevated first in the pleural fluid. Pleural fluid amylase, including salivary amylase, may also be elevated in esophageal rupture and malignancy. Microbiologic and Serologic Tests Microorganisms primarily associated with pleural effusions include Staphylococcus aureus, Enterobacteriaceae, anaerobes, and Mycobacterium tuberculosis. Gram stains, cultures (both aerobic and anaerobic), acid-fast stains, and mycobacteria cultures are performed on pleural fluid when clinically indicated. Serologic testing of pleural fluid is used to differentiate effusions of immunologic origin from noninflammatory processes. Tests for antinuclear antibody (ANA) and rheumatoid factor (RF) are the most frequently performed. Detection of the tumor markers carcinoembryonic antigen (CEA), CA 125 (metastatic uterine cancer), CA 15.3 and CA 549 (breast cancer), and CYFRA 21-1 (lung cancer) provide valuable diagnostic information in effusions of malignant origin.8 Pleural fluid testing and its significance are summarized in Figure 12–15. Pericardial Fluid Normally, only a small amount (10 to 50 mL) of fluid is found between the pericardial serous membranes. Pericardial effusions are primarily the result of changes in the membrane permeability due to infection (pericarditis), malignancy, and trauma-producing exudates. Metabolic disorders such as uremia, hypothyroidism, and autoimmune disorders are the primary causes of transudates. An effusion is suspected when cardiac compression (tamponade) is noted during the physician’s examination (Table 12–8.). PLEURAL FLUID TESTING ALGORITHM Fluid protein and LD Fluid:serum protein >0.5 Fluid:serum LD >0.6 Fluid LD > 2/3 upper normal serum value Appearance Bloody Milky Hematocrit Triglycerides Any positive No Yes Transudate Exudate Glucose ADA WBC/Diff Amylase pH ADA >40 U/L WBC/Diff Amylase pH Lymphocytosis >300/µ L Granulocytosis Elevated <7.2 <6.0 Tuberculosis Bacterial infection Pancreatitis Possible need for chest tubes Esophageal rupture Figure 12–15 Algorithm of pleural fluid testing. 3920_Ch12_229-242 23/01/14 10:35 AM Page 237 Chapter 12 | Serous Fluid Table 12–8 237 Significance of Pericardial Fluid Testing Test Significance Appearance Clear, pale yellow Blood-streaked Grossly bloody Milky Normal, transudate Infection, malignancy Cardiac puncture, anticoagulant medications Chylous and pseudochylous material Additional testing Increased neutrophils Malignant cells Carcinoembryonic antigen Gram stain and culture Acid-fast stain Adenosine deaminase Bacterial endocarditis Metastatic carcinoma Metastatic carcinoma Bacterial endocarditis Tubercular effusion Tubercular effusion Appearance Normal and transudate pericardial fluid appears clear and pale yellow. Effusions resulting from infection and malignancy are turbid, and malignant effusions are frequently blood streaked. Grossly bloody effusions are associated with accidental cardiac puncture and misuse of anticoagulant medications. Milky fluids representing chylous and pseudochylous effusions may also be present. Laboratory Tests Tests performed on pericardial fluid are primarily directed at determining whether the fluid is a transudate or an exudate and include the fluid:serum protein and lactic dehydrogenase (LD) ratios. Like pleural fluid, WBC counts are of little clinical value, although a count of >1000 WBCs/µL with a high percentage of neutrophils can indicate bacterial endocarditis. Cytologic examination of pericardial exudates for the presence of malignant cells is an important part of the fluid analysis. Cells most frequently encountered are the result of metastatic lung or breast carcinoma and resemble those found in pleural fluid. Figure 12–16 shows a metastatic giant mesothelioma cell that is frequently seen in pleural fluid and is associated with asbestos contact. Pericardial fluid tumor marker levels correlate well with cytologic studies.8 Bacterial cultures and Gram stains are performed on concentrated fluids when endocarditis is suspected. Infections are frequently caused by previous respiratory infections including Streptococcus, Staphylococcus, adenovirus, and coxsackievirus. Effusions of tubercular origin are increasing as Figure 12–16 Malignant pericardial effusion showing giant mesothelioma cell with cytoplasmic molding and hyperchromatic nucleoli (×1000). a result of AIDS. Therefore, acid-fast stains and chemical tests for adenosine deaminase are often requested on pericardial effusions. Peritoneal Fluid Accumulation of fluid between the peritoneal membranes is called ascites, and the fluid is commonly referred to as ascitic fluid rather than peritoneal fluid. In addition to the causes of transudative effusions discussed previously, hepatic disorders such as cirrhosis are frequent causes of ascitic transudates. Bacterial infections (peritonitis)—often as a result of intestinal perforation or a ruptured appendix—and malignancy are the most frequent causes of exudative fluids (Table 12–9). Normal saline is sometimes introduced into the peritoneal cavity as a lavage to detect abdominal injuries that have not yet resulted in fluid accumulation. Peritoneal lavage is a sensitive test to detect intra-abdominal bleeding in blunt trauma cases, and results of the RBC count can be used along with radiographic procedures to aid in determining the need for surgery. RBC counts greater than 100,000/mL are indicative of blunt trauma injuries. Cell counts and differentials may also be requested on fluid from peritoneal dialysis to detect infection, and eosinophil counts to detect allergic reactions to the equipment or introduction of air into the peritoneal cavity.1 Transudates Versus Exudates Differentiation between ascitic fluid transudates and exudates is more difficult than for pleural and pericardial effusions. The serum-ascites albumin gradient (SAAG) is recommended over the fluid:serum total protein and LD ratios to detect transudates of hepatic origin.9 Fluid and serum albumin levels are measured concurrently, and the fluid albumin level is then subtracted from the serum albumin level. A difference (gradient) of 1.1 or greater suggests a transudate effusion of hepatic origin, and lower gradients are associated with exudative effusions (see the Example on page 238). 3920_Ch12_229-242 23/01/14 10:35 AM Page 238 238 Part Three | Other Body Fluids Table 12–9 Significance of Peritoneal Fluid Testing Test Appearance Clear, pale yellow Turbid Green Blood-streaked Milky Peritoneal lavage WBC count <500 cells/µL >500 cells/µL Differential Carcinoembryonic antigen CA 125 Glucose Amylase Alkaline phosphatase Significance Normal Microbial infection Bile, gallbladder, pancreatic disorders Trauma, infection, or malignancy Lymphatic trauma and blockage >100,000 RBCs/µL indicates blunt trauma injury Normal Bacterial peritonitis, cirrhosis Bacterial peritonitis, malignancy Malignancy of gastrointestinal origin Malignancy of ovarian origin Decreased in tubercular peritonitis, malignancy Increased in pancreatitis, gastrointestinal perforation Increased in gastrointestinal perforation Ruptured or punctured bladder Laboratory Tests Normal WBC counts are less than 350 cells/µL, and the count increases with bacterial peritonitis and cirrhosis. To distinguish between those two conditions, an absolute neutrophil count should be performed. An absolute neutrophil count >250 cells/µL or >50% of the total WBC count indicates infection. Lymphocytes are the predominant cell in tuberculosis. Cellular Examination Examination of ascitic exudates for the presence of malignant cells is important for detecting tumors of primary and metastatic origin. Malignancies are most frequently of gastrointestinal, prostate, or ovarian origin. Other cells present in ascitic fluid include leukocytes, abundant mesothelial cells, and macrophages, including lipophages (Fig. 12–17). Microorganisms including bacteria, yeast, and Toxoplasma gondii may also be present (Fig. 12–18). Malignant cells of ovarian, prostatic, and colonic origin, often containing mucin-filled vacuoles, are frequently seen (Figs. 12–19 to 12–22). Psammoma bodies containing concentric striations of collagen-like material can be seen in benign conditions and are also associated with ovarian and thyroid malignancies (Fig. 12–23). Blood urea nitrogen/ creatinine Gram stain and culture Bacterial peritonitis Acid-fast stain Tubercular peritonitis Adenosine deaminase Tubercular peritonitis EXAMPLE Figure 12–17 Lipophages (macrophages containing fat droplets) in peritoneal fluid (×500). Serum albumin of 3.8 mg/dL – fluid albumin of 1.2 mg/dL = gradient of 2.6 = transudate effusion Serum albumin of 3.8 mg/dL – fluid albumin of 3.0 mg/dL = gradient of 0.8 = exudate effusion Appearance Like pleural and pericardial fluids, normal peritoneal fluid is clear and pale yellow. Exudates are turbid with bacterial or fungal infections. Green or dark-brown color indicates the presence of bile, which can be confirmed using standard chemical tests for bilirubin. Blood-streaked fluid is seen after trauma and with tuberculosis, intestinal disorders, and malignancy. Chylous or pseudochylous material may be present with trauma or lymphatic vessels blockage. Figure 12–18 Budding yeast in peritoneal fluid (×400). 3920_Ch12_229-242 23/01/14 10:35 AM Page 239 Chapter 12 | Serous Fluid Figure 12–19 Ovarian carcinoma showing community borders, nuclear irregularity, and hyperchromatic nucleoli (×500). Figure 12–20 Ovarian carcinoma cells with large mucin-containing vacuoles (×500). Figure 12–21 Adenocarcinoma of the prostate showing cytoplasmic vacuoles, community borders, and hyperchromatic nucleoli (×500). Chemical Testing Chemical examination of ascitic fluid consists primarily of glucose, amylase, and alkaline phosphatase determinations. Glucose is decreased below serum levels in bacterial and tubercular 239 Figure 12–22 Colon carcinoma cells containing mucin vacuoles and nuclear irregularities (×400). Figure 12–23 Psammoma bodies exhibiting concentric striations (×500). peritonitis and malignancy. Amylase is determined on ascitic fluid to ascertain cases of pancreatitis, and it may be elevated in patients with gastrointestinal perforations. An elevated alkaline phosphatase level is also highly diagnostic of intestinal perforation. Measurements of blood urea nitrogen and creatinine in the fluid are requested when a ruptured bladder or accidental puncture of the bladder during the paracentesis is of concern. Bilirubin is measured when leakage of bile into the peritoneum is suspected following trauma or surgery. Bile contains primarily conjugated bilirubin; therefore, a test for total bilirubin is acceptable.10 Amylase or lipase can be measured to determine whether the pancreatitis or damage to the pancreas is accounting for the accumulation of these pancreatic enzymes in the ascitic fluid.10 Microbiology Tests Gram stains and bacterial cultures for both aerobes and anaerobes are performed when bacterial peritonitis is suspected. Inoculation of fluid into blood culture bottles at the bedside increases the recovery of anaerobic organisms. Acid-fast stains, adenosine deaminase, and cultures for tuberculosis may also be requested. 3920_Ch12_229-242 23/01/14 10:35 AM Page 240 240 Part Three | Other Body Fluids Serologic Tests Measurement of the tumor markers CEA and CA 125 is a valuable procedure for identifying the primary source of tumors producing ascitic exudates. The presence of CA 125 antigen with a negative CEA suggests the source is from the ovaries, fallopian tubes, or endometrium.7 Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Clinical and Laboratory Standards Institute (CLSI): Body Fluid Analysis for Cellular Composition, Approved Guideline C49-A, Wayne, PA, 2006. 2. Jay, SJ: Pleural effusions: Definitive evaluation of the exudate. Postgrad Med 80(5):180–188, 1986. 3. Light, RW: Clinical practice: Pleural effusion. N Engl J Med 346:1971–1977, 2002. 4. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous and Synovial Fluids. A Textbook Atlas. ASCP, Chicago, 1993. 5. Valdez, L, et al: Cholesterol: A useful parameter for distinguishing between pleural exudates and transudates. Chest 99(5): 1097–1102, 1991. 6. Porcel, JM, and Light, RW: Diagnostic approach to pleural fluid effusion in adults. Am Fam Physician 73:1211–1220, 2006. 7. Colice, GL, et al: Medical and surgical treatment of parapneumonic effusions: An evidence-based guideline. Chest 118:1158–1171, 2000. 8. Porcel, JM, et al: Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3 and cytokeratin 19 fragments) in pleural fluid for differential diagnosis of benign and malignant effusions. Chest 126: 1757–1763, 2004. 9. Runyan, BA, et al: The serum-ascites albumin gradient is superior to the exudate transudate concept in the differential diagnosis of ascites. Ann Intern Med 117:215–218, 1992. 10. Clinical and Laboratory Science Institute (CLSI): Analysis of Body Fluids in Clinical Chemistry, Approved Guideline, C49-A, Wayne, PA, 2007, CLSI. Study Questions 1. The primary purpose of serous fluid is to: A. Remove waste products B. Lower capillary pressure C. Lubricate serous membranes D. Nourish serous membranes 2. The membrane that lines the wall of a cavity is the: A. Visceral B. Peritoneal C. Pleural D. Parietal 3. During normal production of serous fluid, the slight excess of fluid is: A. Absorbed by the lymphatic system B. Absorbed through the visceral capillaries C. Stored in the mesothelial cells D. Metabolized by the mesothelial cells 4. Production of serous fluid is controlled by: A. Capillary oncotic pressure B. Capillary hydrostatic pressure C. Capillary permeability D. All of the above 5. An increase in the amount of serous fluid is called a/an: A. Exudate B. Transudate C. Effusion D. Malignancy 6. Pleural fluid is collected by: A. Pleurocentesis B. Paracentesis C. Pericentesis D. Thoracentesis 7. Place the appropriate letter in front of the following statements describing transudates and exudates. A. Transudate B. Exudate ____ Caused by increased hydrostatic pressure ____ Caused by increased capillary permeability ____ Caused by decreased oncotic pressure ____ Caused by congestive heart failure ____ Malignancy related ____ Tuberculosis related ____ Endocarditis related ____ Clear appearance 3920_Ch12_229-242 23/01/14 10:35 AM Page 241 Chapter 12 | Serous Fluid 8. Fluid:serum protein and lactic dehydrogenase ratios are performed on serous fluids: A. When malignancy is suspected B. To classify transudates and exudates C. To determine the type of serous fluid D. When a traumatic tap has occurred 9. Which of the following requires the most additional testing? A. Transudate B. Exudate 10. An additional test performed on pleural fluid to classify the fluid as a transudate or exudate is the: A. WBC count B. RBC count C. Fluid:cholesterol ratio D. Fluid-to-serum protein gradient 11. A milky-appearing pleural fluid indicates: A. Thoracic duct leakage B. Chronic inflammation C. Microbial infection D. Both A and B 12. Which of the following best represents a hemothorax? A. Blood HCT: 42 Fluid HCT: 15 B. Blood HCT: 42 Fluid HCT: 10 C. Blood HCT: 30 Fluid HCT: 10 D. Blood HCT: 30 Fluid HCT: 20 13. All of the following are normal cells seen in pleural fluid except: A. Mesothelial cells B. Neutrophils C. Lymphocytes D. Mesothelioma cells 14. A differential observation of pleural fluid associated with tuberculosis is: A. Increased neutrophils B. Decreased lymphocytes C. Decreased mesothelial cells D. Increased mesothelial cells 15. All of the following are characteristics of malignant cells except: A. Cytoplasmic molding B. Absence of nucleoli C. Mucin-containing vacuoles D. Increased nucleus:cytoplasm ratio 241 16. A pleural fluid pH of 6.0 indicates: A. Esophageal rupture B. Mesothelioma C. Malignancy D. Rheumatoid effusion 17. Plasma cells seen in pleural fluid indicate: A. Bacterial endocarditis B. Primary malignancy C. Metastatic lung malignancy D. Tuberculosis infection 18. A significant cell found in pericardial or pleural fluid that should be referred to cytology is a: A. Reactive lymphocyte B. Mesothelioma cell C. Monocyte D. Mesothelial cell 19. Another name for a peritoneal effusion is: A. Peritonitis B. Lavage C. Ascites D. Cirrhosis 20. A test performed primarily on peritoneal lavage fluid is a/an: A. WBC count B. RBC count C. Absolute neutrophil count D. Amylase 21. The recommended test for determining whether peritoneal fluid is a transudate or an exudate is the: A. Fluid:serum albumin ratio B. Serum ascites albumin gradient C. Fluid:serum lactic dehydrogenase ratio D. Absolute neutrophil count 22. Given the following results, classify this peritoneal fluid: serum albumin, 2.2 g/dL; serum protein, 6.0 g/dL; fluid albumin, 1.6 g/dL. A. Transudate B. Exudate 23. Differentiation between bacterial peritonitis and cirrhosis is done by performing a/an: A. WBC count B. Differential C. Absolute neutrophil count D. Absolute lymphocyte count 3920_Ch12_229-242 23/01/14 10:35 AM Page 242 242 Part Three | Other Body Fluids 24. Detection of the CA 125 tumor marker in peritoneal fluid indicates: A. Colon cancer B. Ovarian cancer C. Gastric malignancy D. Prostate cancer 26. Cultures of peritoneal fluid are incubated: A. Aerobically B. Anaerobically C. At 37°C and 42°C D. Both A and B 25. Chemical tests primarily performed on peritoneal fluid include all of the following except: A. Lactose dehydrogenase B. Glucose C. Alkaline phosphatase D. Amylase Case Studies and Clinical Situations 1. Fluid from a patient with congestive heart failure is collected by thoracentesis and sent to the laboratory for testing. It appears clear and pale yellow and has a WBC count of 450/mL, fluid:serum protein ratio of 0.35, and fluid:serum LD ratio of 0.46. a. What type of fluid was collected? b. Based on the laboratory results, would this fluid be considered a transudate or an exudate? Why? c. List two other tests that could be performed to aid in classifying this fluid. 2. A cloudy pleural fluid has a glucose level of 30 mg/dL (serum glucose level is 100 mg/dL) and a pH of 6.8. a. What condition do these results indicate? b. What additional treatment might the patient receive, based on these results? 3. The following results were obtained on a peritoneal fluid: serum albumin, 2.8 g/dL; fluid albumin, 1.2 g/dL. a. Calculate the SAAG. b. Is this a transudate or an exudate? Why? c. What is the most probable cause of the effusion? 4. Paracentesis is performed on a patient with ascites. The fluid appears turbid and has an elevated WBC count. Additional tests ordered include an absolute granulocyte count, amylase, creatinine, CEA, and CA 125. a. What is the purpose for the absolute granulocyte count? If it is less than 250 cells/mL, what condition is indicated? b. If the amylase level is elevated, what is its significance? State an additional test that might be ordered. c. Explain the significance of an elevated creatinine level. d. What is the purpose of the CEA and CA 125 tests? 5. Describe a situation in which paracentesis might be performed on a patient who does not have ascites. If the RBC count is 300,000/mL, what does this indicate? 6. Microscopic examination of an ascitic fluid shows many cells with nuclear and cytoplasmic irregularities containing psammoma bodies. The CEA test result is normal. What additional test would be helpful? 3920_Ch13_243-254 23/01/14 10:36 AM Page 243 CHAPTER 13 Amniotic Fluid LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 13-1 State the functions of amniotic fluid. 13-2 Describe the formation and composition of amniotic fluid. 13-3 Differentiate maternal urine from amniotic fluid. 13-4 State indications for performing an amniocentesis. 13-5 Describe the specimen-handling and processing procedures for testing amniotic fluid for bilirubin, fetal lung maturity (FLM), and cytogenetic analysis. 13-6 Discuss the principle of the spectrophotometric analysis for evaluation of hemolytic disease of the newborn. 13-7 Interpret a Liley graph. 13-8 Describe the analysis of amniotic fluid for the detection of neural tube disorders. 13-9 Explain the physiologic significance of the lecithinsphingomyelin (L/S) ratio. 13-10 State the relationship of phosphatidyl glycerol to FLM. 13-11 Define lamellar bodies and describe their significance to FLM. 13-12 Discuss the principle of and sources of error for the L/S ratio, Amniostat-FLM, lamellar body count, and Foam Stability Index for FLM. KEY TERMS Amniocentesis Meconium Amnion Hemolytic disease of the newborn (HDN) Amniotic fluid Lamellar bodies Polyhydramnios Cytogenetic analysis Lecithin-sphingomyelin ratio (L/S ratio) Respiratory distress syndrome (RDS) Fetal lung maturity (FLM) Oligohydramnios Surfactants 3920_Ch13_243-254 23/01/14 10:36 AM Page 244 244 Part Three | Other Body Fluids Although the testing of amniotic fluid is frequently associated with cytogenetic analysis, the clinical laboratory also performs several significant tests on amniotic fluid. Because amniotic fluid is a product of fetal metabolism, the constituents that are present in the fluid provide information about the metabolic processes taking place during—as well as the progress of—fetal maturation. When conditions that adversely affect the fetus arise, the danger to the fetus must be measured against the ability of the fetus to survive an early delivery. The tests covered in this chapter are used to determine the extent of fetal distress and fetal maturity (Table 13–1). Physiology Amniotic fluid is present in the amnion, a membranous sac that surrounds the fetus (Fig. 13–1). The amnion is metabolically active and is involved in the exchanges of water and chemicals between the fluid, the fetus, and the maternal circulation; and produces peptides, growth factors, and cytokines. The primary functions of the amniotic fluid are to provide a protective cushion for the fetus, allow fetal movement, stabilize the temperature to protect the fetus from extreme temperature changes, and permit proper lung development. Volume Amniotic fluid volume is regulated by a balance between the production of fetal urine and lung fluid and the absorption Tests for Fetal Well-Being and Maturity Test Reference Values at Term7 Bilirubin scan ⌬ A450 >.025 Alphafetoprotein Lecithinsphingomyelin ratio Amniostatfetal lung maturity Foam Stability Index Optical density 650 nm Lamellar body count <2.0 MoM ≥ 2.0 Significance Hemolytic disease of the newborn Neural tube disorders Fetal lung maturity ≥ 47 Fetal lung maturity/phosphatidyl glycerol Fetal lung maturity ≥ 0.150 Fetal lung maturity ≥ 32,000/mL Fetal lung maturity Positive Chorion Amniotic cavity Umbilical cord Amnion Figure 13–1 Fetus in amniotic sac. Function Table 13–1 Placenta from fetal swallowing and intramembranous flow. Intramembranous flow is the absorption of amniotic fluid water and solutes into the fetal vascular system.1 The amount of amniotic fluid increases in quantity throughout pregnancy, reaching a peak of approximately 800 to 1200 mL during the third trimester, and then gradually decreases prior to delivery. An amniotic fluid volume greater than 1200 mL is called polyhydramnios, whereas amniotic fluid volume less than 800 mL is termed oligohydramnios. In either case, some analytes will be falsely low or falsely high.2 During the first trimester, the approximately 35 mL of amniotic fluid is derived primarily from the maternal circulation. During the latter third to half of pregnancy, the fetus secretes a volume of lung liquid necessary to expand the lungs with growth. During each episode of fetal respiratory movement, secreted lung liquid enters the amniotic fluid, bathing the lungs and washing pulmonary and alveolar contents such as lecithin, sphingomyelin, and phosphatidyl glycerol into the amniotic fluid surrounding the fetus.2 These lung surfactants serve as an index of fetal lung maturity.1 After the first trimester, fetal urine is the major contributor to the amniotic fluid volume. At the time that fetal urine production occurs, fetal swallowing of the amniotic fluid begins and regulates the increase in fluid from the fetal urine. The fetus swallows amniotic fluid, which is absorbed through the gastrointestinal tract and reexcreted by the kidneys from the blood into fetal urine and back into amniotic fluid.2 Failure of the fetus to begin swallowing results in excessive accumulation of amniotic fluid (polyhydramnios) and is an indication of fetal distress, often associated with neural tube disorders. Polyhydramnios may be secondarily associated with fetal structural anomalies, cardiac arrhythmias, congenital infections, or chromosomal abnormalities.1 Increased fetal swallowing, urinary tract deformities, and membrane leakage are possible causes of decreased amniotic fluid (oligohydramnios). Oligohydramnios may be associated with congenital malformations, premature rupture of amniotic membranes, and umbilical cord compression, resulting in decelerated heart rate and fetal death.1 Chemical Composition The placenta is the ultimate source of amniotic fluid water and solutes. Amniotic fluid has a composition similar to that of the 3920_Ch13_243-254 23/01/14 10:36 AM Page 245 Chapter 13 | Amniotic Fluid maternal plasma and contains a small amount of sloughed fetal cells from the skin, digestive system, and urinary tract. These cells provide the basis for cytogenetic analysis. The fluid also contains biochemical substances that are produced by the fetus, such as bilirubin, lipids, enzymes, electrolytes, urea, creatinine, uric acid, proteins, and hormones that can be tested to determine the health or maturity of the fetus. Neural tube defects allow fetal cerebrospinal fluid to enter the amniotic fluid directly. Alpha-fetoprotein and acetylcholinesterase are two biochemical markers tested for these defects.2 A portion of the fluid arises from the fetal respiratory tract, fetal urine, the amniotic membrane, and the umbilical cord. As would be expected, the chemical composition of the amniotic fluid changes when fetal urine production begins. The concentrations of creatinine, urea, and uric acid increase, whereas glucose and protein concentrations decrease. Concentrations of electrolytes, enzymes, hormones, and metabolic end products also vary but are of little clinical significance. Measurement of amniotic fluid creatinine has been used to determine fetal age. Prior to 36 weeks’ gestation, the amniotic fluid creatinine level ranges between 1.5 and 2.0 mg/dL. It then rises above 2.0 mg/dL, thereby providing a means of determining fetal age greater than 36 weeks.3 Differentiating Maternal Urine From Amniotic Fluid Differentiation between amniotic fluid and maternal urine may be necessary to determine possible premature membrane rupture or accidental puncture of the maternal bladder during specimen collection. Chemical analysis of creatinine, urea, glucose, and protein aids in the differentiation. Levels of creatinine and urea are much lower in amniotic fluid than in urine. Creatinine does not exceed 3.5 mg/dL and urea does not exceed 30 mg/dL in amniotic fluid, whereas values as high as 10 mg/dL for creatinine and 300 mg/dL for urea may be found in urine.4 Measurement of glucose and protein by a reagent strip is a less reliable indicator, because glucose and protein are not uncommon urine constituents during pregnancy. However, under normal circumstances, the presence of glucose, protein, or both is associated more closely with amniotic fluid. The fern test also can differentiate amniotic fluid from urine and other body fluids. It is a test used to evaluate premature rupture of the membranes. In the fern test, a vaginal fluid specimen is spread on a glass slide and allowed to completely air dry at room temperature; then it is observed microscopically. The presence of “fern-like” crystals due to the protein and sodium chloride content is a positive screen for amniotic fluid.5 Specimen Collection Indications for Amniocentesis Amniocentesis is recommended for neural tube defects when screening blood tests such as the maternal serum alphafetoprotein test are abnormal or to detect genetic disorders or 245 to evaluate the health of the fetus. Fetal body measurements taken with ultrasonography accurately estimate the gestational age of the fetus and provide an assessment of the size and growth of the fetus throughout pregnancy to diagnose and manage intrauterine growth retardation. Finding an abnormality on the ultrasound could indicate potential fetal development problems and indicate the need for an amniocentesis and laboratory measurements of fetal lung maturity. Fetal epithelial cells in amniotic fluid indicate the genetic material of the fetus and the biochemical substances that the fetus has produced. These cells can be separated from the fluid, cultured, and examined for chromosome abnormalities by karyotyping, fluorescence in situ hybridization (FISH), fluorescent mapping spectral karyotyping (SKY), and DNA testing. Biochemical substances produced by the fetus can be analyzed by thin-layer chromatography to evaluate the health of the fetus. (Table 13–2) In general, amniocentesis is a safe procedure, particularly when performed after the 14th week of gestation. Fluid for chromosome analysis is usually collected at approximately 16 weeks’ gestation and tests for intrauterine growth retardation are performed near the end of the second trimester, whereas tests for fetal distress and maturity are performed later in the third trimester.2 Table 13–2 Indications for Performing Amniocentesis Amniocentesis may be indicated at 15 to 18 weeks’ gestation for the following conditions to determine early treatment or intervention: • Mother’s age of 35 or older at delivery • Family history of chromosome abnormalities, such as trisomy 21 (Down syndrome) • Parents carry an abnormal chromosome rearrangement • Earlier pregnancy or child with birth defect • Parent is a carrier of a metabolic disorder • Family history of genetic diseases such a sickle cell disease, Tay-Sachs disease, hemophilia, muscular dystrophy, sickle cell anemia, Huntington chorea, and cystic fibrosis • Elevated maternal serum alpha-fetoprotein • Abnormal triple marker screening test • Previous child with a neural tube disorder such as spina bifida, or ventral wall defects (gastroschisis) • Three or more miscarriages Amniocentesis is indicated later in the pregnancy (20 to 42 weeks) to evaluate: • Fetal lung maturity • Fetal distress • HDN caused by Rh blood type incompatibility • Infection 3920_Ch13_243-254 23/01/14 10:36 AM Page 246 246 Part Three | Other Body Fluids Collection Amniotic fluid is obtained by needle aspiration into the amniotic sac, a procedure called amniocentesis. The procedure most frequently performed is a transabdominal amniocentesis. Using continuous ultrasound for guidance, the physician locates the fetus and placenta to safely perform the procedure. A thin, hollow needle is inserted through the mother’s abdomen into the mother’s uterus and into the amniotic sac to aspirate the amniotic fluid. Vaginal amniocentesis may also be performed; however, this method carries a greater risk of infection. A maximum of 30 mL of amniotic fluid is collected in sterile syringes. The first 2 or 3 mL collected can be contaminated by maternal blood, tissue fluid, and cells and are discarded. Specimens should be transferred to sterile plastic containers and taken immediately to the laboratory. Fluid for bilirubin analysis in cases of hemolytic disease of the newborn (HDN) must be protected from light at all times. Specimen Handling and Processing Handling and processing of amniotic fluid vary with the tests requested and with the methodology used by the laboratory performing the test. However, in all circumstances, special handling procedures should be performed immediately and the specimen delivered promptly to the laboratory. Fluid for fetal lung maturity (FLM) tests should be placed in ice for delivery to the laboratory and kept refrigerated. Specimens for bilirubin testing must be immediately protected from light. This can be accomplished by placing the specimens in amber-colored tubes, wrapping the collection tube in foil, or by use of a black plastic cover for the specimen container. Specimens for cytogenetic studies or microbial studies must be processed aseptically and maintained at room temperature or body temperature (37°C incubation) prior to analysis to prolong the life of the cells needed for analysis. All fluid for chemical testing should be separated from cellular elements and debris as soon as possible to prevent distortion of chemical constituents by cellular metabolism or disintegration. This can be performed using centrifugation or filtration. The time and speed of centrifugation also depends on the test and lab protocol. Color and Appearance Normal amniotic fluid is colorless and may exhibit slight to moderate turbidity from cellular debris, particularly in later stages of fetal development. Blood-streaked fluid may be present as the result of a traumatic tap, abdominal trauma, or intraamniotic hemorrhage. The source of the blood (maternal or fetal) can be determined using the Kleihauer-Betke test for fetal hemoglobin and is important for further case management. The presence of bilirubin gives the fluid a yellow color and is indicative of red blood cell destruction resulting from HDN. Meconium, which is usually defined as a newborn’s first bowel movement, is formed in the intestine from fetal intestinal secretions and swallowed amniotic fluid. It is a dark green, mucus-like material. It may be present in the amniotic fluid as a result of fetal distress. Fetal aspiration of meconium during fetal swallowing is a concern when increased amounts are present in the fluid. A very dark red-brown fluid is associated with fetal death (Table 13–3). Tests for Fetal Distress Hemolytic Disease of the Newborn The oldest routinely performed laboratory test on amniotic fluid evaluates the severity of the fetal anemia produced by HDN. The incidence of this disease has been decreasing rapidly since the development of methods to prevent anti-Rh antibody production in postpartum mothers. However, antibodies against other red cell antigens are also capable of producing HDN, and immunization of Rh-negative mothers may not be effective or even performed in all cases. Initial exposure to foreign red cell antigens occurs during gestation, delivery of the placenta, or a previous pregnancy when fetal red blood cells enter into the maternal circulation and stimulate the mother to produce antibodies to the antigen. When these antibodies present in the maternal circulation cross the placenta into the fetal circulation and bind to the antigen on the fetal cells, the cells are destroyed. The destruction of fetal red blood cells results in the appearance of the red blood cell degradation product, unconjugated bilirubin, in the amniotic fluid. By measuring the amount of bilirubin in the fluid, the extent of hemolysis taking place may be determined, and the danger this anemia presents to the fetus may be assessed (Fig. 13–2). Amniotic fluid bilirubin is measured by spectrophotometric analysis using serial dilutions. As illustrated in Figure 13–3, the optical density (OD) of the fluid is measured in intervals between 365 nm and 550 nm and the readings plotted on semilogarithmic graph paper. In normal fluid, the OD is highest at 365 nm and decreases linearly to 550 nm, illustrated by a straight line. When bilirubin is present, a rise in OD is seen at 450 nm because this is the wavelength of maximum bilirubin absorption. The difference between the OD of the theoretic baseline and the OD at 450 nm represents the amniotic fluid Table 13–3 Amniotic Fluid Color Color Significance Colorless Blood-streaked Normal Traumatic tap, abdominal trauma, intra-amniotic hemorrhage Hemolytic disease of the newborn (bilirubin) Meconium Fetal death Yellow Dark green Dark red-brown 3920_Ch13_243-254 23/01/14 10:36 AM Page 247 Chapter 13 | Amniotic Fluid Baby 1 Figure 13–2 Rh antibodies crossing the placenta. 0.6 Absorbance 0.5 Rh+ Rh+ fetus Fetal blood enters maternal circulation via placenta IgG antibodies cross placenta to destroy fetal Rh+ RBCs IgG antibodies remain in mother’s circulation 0.1 410 450 500 Rh+ Rh+ fetus No rm sca al n 365 Baby 2 2nd Pregnancy 0.4 0.2 Rh antibody 1st Pregnancy Oxyhemoglobin Bilirubin peak at 410 peak at 450 0.3 Rh– Rh– Rh– 247 550 Wavelength (nm) Figure 13–3 Spectrophotometric bilirubin scan showing bilirubin and oxyhemoglobin peaks. bilirubin concentration. This difference in OD, referred to as the absorbance difference at 450 nm (⌬ A450), is then plotted on a Liley graph to determine the severity of the hemolytic disease (Fig. 13–4).6 Notice that the Liley graph plots the ⌬ A450 against gestational age and is divided into three zones that represent the extent of hemolytic severity. Values falling in zone I indicate no more than a mildly affected fetus; those in zone II indicate moderate hemolysis and require careful monitoring anticipating an early delivery or exchange transfusion upon delivery, whereas a value in zone III indicates severe hemolysis and suggests a severely affected fetus. Intervention through induction of labor or intrauterine exchange transfusion must be considered when a ⌬ A450 is plotted in zone III. As mentioned, specimens must be protected from light at all times. Markedly decreased values will be obtained with as little as 30 minutes of exposure to light. Contamination of the fluid by cells, hemoglobin, meconium, or other debris will interfere with the spectrophotometric analysis. Specimens should be immediately centrifuged to remove particulate interference. Specimens contaminated with meconium will cause falsely low ⌬ A450 values and are not acceptable for spectrophotometric analysis. Specimens that are contaminated with blood are generally unacceptable because maximum absorbance of oxyhemoglobin occurs at 410 nm and can interfere with the bilirubin absorption peak (see Fig. 13–3). This interference can be removed by extraction with chloroform if necessary.7 A control may be prepared by diluting commercial chemistry control sera 1 to 10 with normal saline and treating it in the same manner as the patient specimen. Bilirubin and protein levels approximate those in amniotic fluid and can be varied by using low or high control sera.8 Neural Tube Defects Neural tube defects (NTD) are one of the most common birth defects in the United States. It can be detected by maternal serum alpha-fetoprotein (MSAFP) blood test, high-resolution ultrasound, and amniocentesis. Increased levels of alpha-fetoprotein (AFP) in both the maternal circulation and the amniotic fluid can be indicative of fetal neural tube defects, such as anencephaly and spina bifida. AFP is the major protein produced by the fetal liver during early gestation (prior to 18 weeks). It is found in the maternal serum due to the combined fetal-maternal circulations and in the amniotic fluid from diffusion and excretion of fetal urine. Increased levels are found in the maternal 3920_Ch13_243-254 23/01/14 10:36 AM Page 248 248 Part Three | Other Body Fluids 1.00 0.90 0.80 0.70 0.60 0.50 ∆ O.D. Change in optical density 0.40 0.30 Severely affected fetus intervention required Zone III 0.20 Zone II 0.10 0.09 0.08 0.07 Moderately affected fetus requiring close monitoring 0.06 0.05 0.04 0.03 Zone I 0.02 Nonaffected or mildly affected fetus 0.01 20 22 24 26 28 30 32 34 Gestational age in weeks serum and amniotic fluid when the skin fails to close over the neural tissue, as occurs in anencephaly and spina bifida. Measurement of amniotic fluid AFP levels is indicated when maternal serum levels are elevated or a family history of previous neural tube defects exists. The possibility of a multiple pregnancy also must be investigated when serum levels are elevated. Normal values are based on the week of gestational age, as the fetus produces maximal AFP between 12 and 15 weeks’ gestation, after which levels in amniotic fluid begin to decline. Both serum and amniotic fluid AFP levels are reported in terms of multiples of the median (MoM). The median is the laboratory’s reference level for a given week of gestation. A value two times the median value is considered abnormal (greater than 2 MoM) for both maternal serum and amniotic fluid. Elevated amniotic fluid AFP levels are followed by measurement of amniotic acetylcholinesterase (AChE). The test is more specific for neural tube disorders than AFP, provided it is not performed on a bloody specimen, because blood contains AChE.4 Tests for Fetal Maturity Fetal distress, whether caused by HDN or other conditions, forces the obstetrician to consider a preterm delivery. At this point, fetal maturity must be assessed. Fetal Lung Maturity Respiratory distress syndrome (RDS) is the most frequent complication of early delivery and is the seventh most common cause of morbidity and mortality in the premature infant.9 This disease is caused by an insufficiency of lung surfactant 36 38 40 Figure 13–4 Example of a Liley graph. production and structural immaturity of the fetal lungs. Surfactant normally appears in mature lungs and allows the alveoli (air sacs of the lung) to remain open throughout the normal cycle of inhalation and exhalation. Surfactant keeps the alveoli from collapsing by decreasing surface tension and allows them to inflate with air more easily. If the surfactant concentrations are too low, the alveoli will collapse, causing RDS. The incidence of RDS decreases with increasing gestational age and lung maturity.9 Therefore, laboratory tests must be performed to determine the maturity of the fetal lungs. The amount of surfactant in fetal lungs can be estimated by measuring the amount of surfactants in amniotic fluid.9 Several laboratory tests are available to measure FLM. Lecithin-Sphingomyelin Ratio The reference method to which tests of FLM are compared is the lecithin-sphingomyelin (L/S) ratio. Lecithin is the primary component of the surfactants (phospholipids, neutral lipids, and proteins) that make up the alveolar lining and account for alveolar stability. Lecithin is produced at a relatively low and constant rate until the 35th week of gestation, at which time a noticeable increase in its production occurs, resulting in the stabilization of the fetal lung alveoli. Sphingomyelin is a lipid that is produced at a constant rate after about 26 weeks’ gestation; therefore, it can serve as a control on which to base the rise in lecithin. Both lecithin and sphingomyelin appear in the amniotic fluid in amounts proportional to their concentrations in the fetus.10 Prior to 35 weeks’ gestation, the L/S ratio is usually less than 1.6 because large amounts of lecithin are not being produced at this time. After 35 weeks’ gestation, the 3920_Ch13_243-254 23/01/14 10:37 AM Page 249 Chapter 13 | Amniotic Fluid lecithin concentration increases while the sphingomyelin concentration remains constant. The L/S ratio will rise to 2.0 or higher as the lecithin production increases to prevent alveolar collapse. Therefore, when the L/S ratio reaches 2.0, a preterm delivery is usually considered to be a relatively safe procedure. Falsely elevated results are encountered in fluid contaminated with blood or meconium because both these substances contain lecithin and sphingomyelin. Quantitative measurement of lecithin and sphingomyelin is performed using thin-layer chromatography (TLC). Because the procedure is labor intensive and subject to high coefficients of variation, many laboratories have replaced the L/S ratio with the quantitative phosphatidyl glycerol immunoassays and lamellar body density procedures.11 Phosphatidyl Glycerol The presence of another lung surface lipid, phosphatidyl glycerol (PG), is also essential for adequate lung maturity and can be detected after 35 weeks’ gestation. The production of PG normally parallels that of lecithin, but its production is delayed in cases of maternal diabetes. In this circumstance, respiratory distress occurs in the presence of an L/S ratio of 2.0. Therefore, a thin-layer chromatography lung profile must include lecithin, sphingomyelin, and PG to provide an accurate measurement of FLM.12 Development of an immunologic agglutination test for PG has provided a more rapid and easy to perform method for assessment of fetal maturity that does not require a laboratory to be equipped to perform thin-layer chromatography. The Aminostat-FLM (Irving Scientific, Santa Ana, CA) uses antisera containing polyclonal anti-PG antibodies that are specific for PG-containing lamellar bodies in the amniotic fluid. The size of the agglutinates is read macroscopically and the results are reported as either negative, indicating pulmonary immaturity, or low positive or high positive, indicating pulmonary maturity. The test is not affected by specimen contamination with blood and meconium.13 Studies have shown good correlation with thin-layer chromatography but with a slightly higher incidence of false-negative results that may need to be followed up with further testing.14,15 gradient of ethanol/fluid ratios ranging from 0.42 mL to 0.55 mL in 0.01-mL increments, which can be used to provide a semiquantitative measure of the amount of surfactant present. A value of 47 or higher indicates FLM. The Foam Stability Index has shown good correlation with the L/S ratio and tests for phosphatidyl glycerol. The test cannot be used with contaminated amniotic fluid because blood and meconium also reduce surface tension, yielding a falsely mature index result. Lamellar Bodies Surfactant is composed of approximately 90% phospholipid and 10% protein and is packaged into layered storage granules called lamellar bodies.9,18 Lamellar bodies are densely packed layers of phospholipids that represent a storage form of pulmonary surfactant. They are secreted by the type II pneumocytes of the fetal lung at about 24 weeks of gestation and are absorbed into the alveolar spaces to provide surfactant. They enter the amniotic fluid at about 26 weeks of gestation and increase in concentration from 50,000 to 200,000 per microliter by the end of the third trimester. As the fetal lung matures, increased lamellar body production is reflected by an increase in amniotic fluid phospholipids and the L/S ratio.19 Therefore, the number of lamellar bodies present in the amniotic fluid correlates with the amount of phospholipid present in the fetal lungs. The presence of lamellar bodies increases the OD of the amniotic fluid. Specimens are centrifuged at 2000 g for 10 minutes and examined using a wavelength of 650 nm, which rules out interference from hemoglobin but not other PROCEDURE 13-1 Foam Shake Test 1. 2. 3. 4. Mix equal parts of amniotic fluid with 95% ethanol. Vigorously shake for 15 seconds. Allow to sit undisturbed for 15 minutes. Observe for the presence of a continuous line of bubbles around the outside edge. Foam Stability Index Until the development of biochemical techniques to measure the individual lung-surface lipid concentrations, a mechanical screening test, called the “foam” or “shake” test, was used to determine their presence. Because it can be performed at the bedside or in the laboratory, the test is still in use. Amniotic fluid is mixed with 95% ethanol, shaken for 15 seconds, and allowed to sit undisturbed for 15 minutes. At the end of this time, the surface of the fluid is observed for the presence of a continuous line of bubbles around the outside edge. The presence of bubbles indicates that a sufficient amount of phospholipid is available to reduce the surface tension of the fluid even in the presence of alcohol, an antifoaming agent. A modification of the foam test uses 0.5 mL of amniotic fluid added to increasing amounts of 95% ethanol, providing a 249 PROCEDURE 13-2 Foam Stability Index 1. Add 0.5 mL of amniotic fluid to tubes containing increasing amounts of 95% ethanol ranging from 0.42 to 0.55 mL in 0.01-mL increments. 2. Vigorously shake for 15 seconds. 3. Allow to sit undisturbed for 15 minutes. 4. Observe for the presence of a continuous line of bubbles around the outside edge. 5. Values ≥ 47 indicate fetal lung maturity. 3920_Ch13_243-254 23/01/14 10:37 AM Page 250 250 Part Three | Other Body Fluids HISTORICAL NOTE Microviscosity: Fluorescence Polarization Assay The fluorescence polarization assay is no longer performed because the manufacturer (Abbott Laboratories, Abbott Park, IL) discontinued the instrument and reagents in 2011. It was the most widely used test in evaluating FLM and was considered an excellent screening tool for FLM because the results correlated well with an L/S ratio of 2.0.16 The fluorescence polarization assay for FLM measured the surfactant to albumin (S/A) ratio to measure FLM. The test was based on the principle that phospholipids decrease the microviscosity of the amniotic fluid. This change in microviscosity was measured using the principle of fluorescence polarization using the TDX/TDxFLx FLM II Assay System (Abbott Laboratories, Abbott Park, IL). The assay measured the polarization of a fluorescent dye that combined with both surfactants and albumin in the amniotic fluid. The fluorescent dye bound to surfactant had a longer fluorescence lifetime and a low polarization, whereas dye bound to albumin had a decreased fluorescence lifetime and a high polarization. The recorded changes in polarization produced a surfactant/albumin ratio expressed in milligrams surfactant to grams albumin that was compared with a fetal lung maturity II assay calibration standard curve using calibrators of known surfactant/albumin content. A surfactant/albumin ratio of 55 mg/g indicated FLM, whereas values between 40 mg/g and 54 mg/g were indeterminate and ratios less than or equal to 39 mg/g indicated immature lungs.17 The test required 1.0 mL of amniotic fluid, and specimens contaminated with blood, meconium, suspected maternal urine, and visibly icteric sample could not be used. contaminants, such as meconium. An OD of 0.150 has been shown to correlate well with an L/S ratio of greater than or equal to 2.0 and the presence of phosphatidyl glycerol.20 volume of amniotic fluid required, and (6) excellent clinical performance.18 Amniotic fluid specimens containing whole blood, meconium, and mucus should not be used. Blood initially raises the LBC because of the presence of platelets and then can lower the LBC as lamellar bodies are trapped in the fibrin strands.18 Meconium and mucus cause a false increase in the LBC and should not be used for testing. Specimens may be stored at 2°C to 8°C but never frozen. A consensus protocol for performing LBC has been published by CLSI and is described in Procedure 13–3. Results are reported in units of lamellar bodies per microliter and should be accompanied by the laboratory’s established values for maturity and the instrument that was used. A consensus protocol for noncentrifuged samples considers LBCs greater than 50,000/L an indication of FLM and values below 15,000/L as immature. Results in between these two values are considered indeterminate and further testing using alternate methods is recommended.21 PROCEDURE 13-3 Lamellar Body Count18 1. Mix the amniotic fluid sample by inverting the capped sample container five times. 2. Transfer the fluid to a clear test tube to allow for visual inspection. 3. Visually inspect the specimen. Fluids containing obvious mucus, whole blood, or meconium should not be processed for an LBC. 4. Cap the tube and mix the sample by gentle inversion or by placing the test tube on a tube rocker for 2 minutes. 5. Flush the platelet channel; analyze the instrument’s diluents buffer until a background count deemed acceptable by the laboratory is obtained in two consecutive analyses. 6. Process the specimen through the cell counter and record the platelet channel as the LBC. Log on to www.fadavis.com/strasinger for additional content related to this chapter. Lamellar Body Count Lamellar body diameter is similar to that of small platelets ranging in size from 1.7 to 7.3 fL, or 1 to 5m; therefore, lamellar body counts (LBCs) can be obtained using the platelet channel of automated hematology analyzers using either optical or impedance methods for counting.18 Some automated cell counters use both impedance and optical methods for counting. Because the various hematology analyzers count lamellar bodies differently and require different specimen preparation, cutoff values for FLM vary, making it necessary to establish analyzer-specific LBC clinical decision limits.18 The advantages of lamellar body counting include: (1) rapid turnaround time, (2) low reagent cost, (3) wide availability, (4) low degree of technical difficulty, (5) low References 1. Ross, MG, Brace, RA, and the NIH Workshop Participants, National Institute of Child Health and Development Conference summary: Amniotic fluid biology—basic and clinical aspects. J Maternal-Fetal Med 10:2–19, 2001. 2. Clinical and Laboratory Standards Institute (CLSI): Analysis of Body Fluids in Clinical Chemistry: approved guideline, CLSI Document C49-A, Wayne, PA, 2007, CLSI. 3. Weiss, RR, et al: Amniotic fluid uric acid and creatinine as measures of fetal maturity. Obstet Gynecol 44(2):208–214, 1974. 3920_Ch13_243-254 23/01/14 10:37 AM Page 251 Chapter 13 | Amniotic Fluid 4. Wenk, RE, and Rosenbaum, JM: Examination of amniotic fluid. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 1996. 5. Heron, HJ: The use of the Fern test to differentiate amniotic fluid from urine. Triangle Jul:20:60–62, 1963. 6. Liley, AW: Liquor amnii analysis in the management of the pregnancy complicated by Rhesus sensitization. Am J Obstet Gynecol 82:1359, 1961. 7. Spinnato, JA, et al: Amniotic bilirubin and fetal hemolytic disease. Am J Obstet Gynecol 165(4):1030–1035, 1991. 8. McDonald, OL, and Watts, MT: Use of commercially prepared control sera as quality control materials for spectrophotometric bilirubin determinations in amniotic fluid. Am J Clin Pathol 84(4):513–517, 1985. 9. Grenache, D: Fetal lung maturity testing: what labs need to know now. Medical Lab Observer (MLO), February 2012. 10. Gluck, L, et al: Diagnosis of the respiratory distress syndrome by amniocentesis. Am J Obstet Gynecol 109(3):440–445, 1971. 11. Dubin, SB: Assessment of FLM: Practice parameter. Am J Clin Pathol 110:723–732, 1998. 12. Kulovich, MV, Hallman, MB, and Gluck, L: The lung profile: Normal pregnancy. Am J Obstet Gynecol 135:57–60, 1979. 13. Eisenbrey, AB, et al: Phosphatidyl glycerol in amniotic fluid: Comparison of an “ultrasensitive” immunologic assay with TLC and enzymatic assay. Am J Clin Pathol 91(3):293–297, 1989. 251 14. Chapman, JF: Current methods for evaluating FLM. Lab Med 17(10):597–602, 1986. 15. Saad, SA, et al: The reliability and clinical use of a rapid phosphatidyl glycerol assay in normal and diabetic pregnancies. Am J Obstet Gynecol 157(6):1516–1520, 1987. 16. Winn-McMillan, T, and Karon, BS: Comparison of the TDx-FLM II and lecithin to sphingomyelin ratio assays in predicting fetal lung maturity. Am J Obstet Gynecol 193,778–782, 2005. 17. TDx/TDxFLx Fetal Lung Maturity II (FLM II) package insert. Reference 7A76. Abbott Laboratories, Diagnostics Division, Abbott Park, IL, April 2003. 18. Clinical and Laboratory Standards Institute. Assessment of fetal lung maturity by the lamellar body count; approved guideline, CLSI document C58-A. Wayne, PA, CLSI, 2011. 19. Khazardoost, S, et al: Amniotic fluid lamellar body count and its sensitivity and specificity in evaluating of fetal lung maturity. J Obstet Gynaecol 25(3):257–259, 2005. 20. Sbarra, AJ, et al: Correlation of amniotic fluid optical density at 650 nm and lecithin/sphingomyelin ratios. Obstet Gynecol 48:613, 1976. 21. Lu Ji, Gronowski, AM, Eby, C,: Lamellar Body Counts Performed on Automated Hematology Analyzers to Assess Fetal Lung Maturity. LabMedicine 39(7): 419–423, 2008. Study Questions 1. Which of the following is not a function of amniotic fluid? A. Allows movement of the fetus B. Allows carbon dioxide and oxygen exchange C. Protects fetus from extreme temperature changes D. Acts as a protective cushion for the fetus 2. What is the primary cause of the normal increase in amniotic fluid as a pregnancy progresses? A. Fetal cell metabolism B. Fetal swallowing C. Fetal urine D. Transfer of water across the placenta 3. Which of the following is not a reason for decreased amounts of amniotic fluid? A. Fetal failure to begin swallowing B. Increased fetal swallowing C. Membrane leakage D. Urinary tract defects 4. Why might a creatinine level be requested on an amniotic fluid? A. Detect oligohydramnios 5. Amniotic fluid specimens are placed in amber-colored tubes prior to sending them to the laboratory to prevent the destruction of: A. Alpha-fetoprotein B. Bilirubin C. Cells for cytogenetics D. Lecithin 6. How are specimens for FLM testing delivered to and stored in the laboratory? A. Delivered on ice and refrigerated B. Immediately centrifuged C. Kept at room temperature D. Delivered in a vacuum tube 7. Why are amniotic specimens for cytogenetic analysis incubated at 37°C prior to analysis? A. To detect the presence of meconium B. To differentiate amniotic fluid from urine C. To prevent photo-oxidation of bilirubin to biliverdin D. To prolong fetal cell viability and integrity 8. Match the following colors in amniotic fluid with their significance. B. Detect polyhydramnios ___ A. Colorless 1. Fetal death C. Differentiate amniotic fluid from maternal urine ___ B. Dark green 2. Normal ___ C. Red-brown 3. Presence of bilirubin ___ D. Yellow 4. Presence of meconium D. Evaluate lung maturity 3920_Ch13_243-254 23/01/14 10:37 AM Page 252 252 Part Three | Other Body Fluids 9. A significant rise in the OD of amniotic fluid at 450 nm indicates the presence of which analyte? A. Bilirubin B. Lecithin C. Oxyhemoglobin D. Sphingomyelin 15. When performing an L/S ratio by thin-layer chromatography, a mature fetal lung will show: A. Sphingomyelin twice as concentrated as lecithin B. No sphingomyelin C. Lecithin twice as concentrated as sphingomyelin D. Equal concentrations of lecithin and sphingomyelin 10. Plotting the amniotic fluid OD on a Liley graph represents the severity of hemolytic disease of the newborn. A value that is plotted in zone II indicates what condition of the fetus? A. No hemolysis B. Mildly affected fetus C. Moderately affected fetus that requires close monitoring D. Severely affected fetus that requires intervention 16. True or False: Phosphatidyl glycerol is present with an L/S ratio of 1.1. 11. The presence of a fetal neural tube disorder may be detected by: A. Increased amniotic fluid bilirubin B. Increased maternal serum alpha-fetoprotein C. Decreased amniotic fluid phosphatidyl glycerol D. Decreased maternal serum acetylcholinesterase 12. True or False: An AFP MoM value greater than two times the median value is considered an indication of a neural tube disorder. 13. When severe HDN is present, which of the following tests on the amniotic fluid would the physician not order to determine whether the fetal lungs are mature enough to withstand a premature delivery? A. AFP levels B. Foam stability index C. Lecithin/sphingomyelin ratio D. Phosphatidyl glycerol detection 17. A rapid immunologic test for FLM that does not require performance of thin-layer chromatography is: A. AFP levels B. Amniotic acetylcholinesterase C. Aminostat-FLM D. Bilirubin scan 18. Does the failure to produce bubbles in the Foam Stability Index indicate increased or decreased lecithin? A. Increased B. Decreased 19. The presence of phosphatidyl glycerol in amniotic fluid fetal lung maturity tests must be confirmed when: A. Hemolytic disease of the newborn is present B. The mother has maternal diabetes C. Amniotic fluid is contaminated by hemoglobin D. Neural tube disorder is suspected 20. A lamellar body count of 50,000 correlates with: A. Absent phosphatidyl glycerol and L/S ratio of 1.0 B. L/S ratio of 1.5 and absent phosphatidyl glycerol C. OD at 650 nm of 1.010 and an L/S ratio of 1.1 D. OD at 650 nm of 0.150 and an L/S ratio of 2.0 14. True or False: Prior to 35 weeks’ gestation, the normal L/S ratio is less than 1.6. Case Studies and Clinical Situations 1. Amniocentesis is performed on a woman believed to be in approximately the 31st week of gestation. This is the second pregnancy for this Rh-negative woman with diabetes. Spectrophotometric analysis of the fluid shows a ∆ A450 of 0.3. a. Based on the Liley graph, should the physician consider inducing labor? b. What else must the physician consider prior to inducing labor? The physician decides to induce labor based on a positive Aminostat-FLM. c. What information did this test provide for the physician? d. Why did the physician prefer an Aminostat-FLM to an L/S ratio in this situation? 2. Amniocentesis is performed following a maternal serum AFP level of 2.2 MoM at 15 weeks’ gestation. a. What fetal condition is suspected? b. If the amniotic fluid AFP is 2.5 MoM, what additional test could be performed? c. In what situation would this additional test not be performed? 3920_Ch13_243-254 23/01/14 10:37 AM Page 253 Chapter 13 | Amniotic Fluid 3. How might a dark green amniotic fluid affect the results of the following tests? a. Foam Stability Index b. L/S ratio c. Aminostat-FLM d. OD650 4. How might a blood-streaked amniotic fluid affect the results of the following tests? a. L/S ratio b. AChE c. Bilirubin analysis d. Aminostat-FLM 253 5. Amniocentesis is performed on a woman whose last two pregnancies resulted in stillbirths due to hemolytic disease of the newborn. A screening test performed at the hospital is positive for bilirubin, and the specimen is sent to a reference laboratory for a bilirubin scan. Physicians are concerned when the report comes back negative. What factors would be considered in evaluating this result. a. Correct specimen was sent b. Specimen was refrigerated c. Specimen was exposed to light d. Specimen reached the reference lab within 30 mins 3920_Ch13_243-254 23/01/14 10:37 AM Page 254 3920_Ch14_255-268 23/01/14 10:38 AM Page 255 CHAPTER 14 Fecal Analysis LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 14-1 Describe the normal composition and formation of feces. 14-10 State the significance of increased neutrophils in a stool specimen. 14-2 Differentiate between secretory and osmotic diarrhea using fecal electrolytes, fecal osmolality, and stool pH. 14-11 Describe a positive microscopic examination for muscle fibers. 14-3 List three causes of secretory and osmotic diarrhea. 14-12 Name the fecal fats stained by Sudan III, and give the conditions under which they will stain. 14-4 Describe the mechanism of altered motility and at least three conditions that can cause it. 14-5 List three causes of steatorrhea. 14-6 Differentiate malabsorption from maldigestion syndromes and name a test that distinguishes the two conditions. 14-7 Instruct patients in the collection of random and quantitative stool specimens. 14-8 State a pathogenic and a nonpathogenic cause for stools that are red, black, and pale yellow. 14-9 State the significance of bulky, ribbon-like, and mucus-containing stools. 14-13 Describe and interpret the microscopic results that are seen when a specimen from a patient with steatorrhea is stained with Sudan III. 14-14 Discuss the collection procedure for a quantitative fecal fat and methods for analysis. 14-15 Explain the methods used to detect fecal occult blood. 14-16 Instruct a patient in the collection of specimens for occult blood, including an explanation of dietary restrictions for the guaiac test. 14-17 Briefly describe a chemical screening test performed on feces for each of the following: fetal hemoglobin, pancreatic insufficiency, and carbohydrate intolerance. KEY TERMS Acholic stools Malabsorption Osmotic diarrhea Constipation Maldigestion Secretory diarrhea Diarrhea Occult blood Steatorrhea 3920_Ch14_255-268 23/01/14 10:38 AM Page 256 256 Part Three | Other Body Fluids In the minds of most laboratory personnel, fecal specimen analysis fits into the category of a “necessary evil.” However, as an end product of body metabolism, feces do provide valuable diagnostic information. Routine fecal examination includes macroscopic, microscopic, and chemical analyses for the early detection of gastrointestinal (GI) bleeding, liver and biliary duct disorders, maldigestion/malabsorption syndromes, pancreatic diseases, inflammation, and causes of diarrhea and steatorrhea. Of equal diagnostic value is the detection and identification of pathogenic bacteria, viruses, and parasites; however, these procedures are best covered in a microbiology textbook and are not discussed here. Physiology The normal fecal specimen contains bacteria, cellulose, undigested foodstuffs, GI secretions, bile pigments, cells from the intestinal walls, electrolytes, and water. Approximately 100 to 200 g of feces is excreted in a 24-hour period. Many species of bacteria make up the normal flora of the intestines. Bacterial metabolism produces the strong odor associated with feces and intestinal gas (flatus). Carbohydrates, especially oligosaccharides, that are resistant to digestion pass through the upper intestine unchanged but are metabolized by bacteria in the lower intestine, producing large amounts of flatus. Excessive gas production also occurs in lactoseintolerant people when the intestinal bacteria metabolize the lactose from consumed milk or lactose-containing substances. Although digestion of ingested proteins, carbohydrates, and fats takes place throughout the alimentary tract, the small intestine is the primary site for the final breakdown and reabsorption of these compounds. Digestive enzymes secreted into the small intestine by the pancreas include trypsin, chymotrypsin, amino peptidase, and lipase. Bile salts provided by the liver aid in the digestion of fats. A deficiency in any of these substances creates an inability to digest and, therefore, to reabsorb certain foods. Excess undigested or unreabsorbed materials then appear in the feces, and the patient exhibits symptoms of maldigestion and malabsorption. As shown in Figure 14–1, approximately 9000 mL of ingested fluid, saliva, gastric, liver, pancreatic, and intestinal secretions enter the digestive tract each day. Under normal conditions, only between 500 to 1500 mL of this fluid reaches the large intestine, and only about 150 mL is excreted in the feces. Water and electrolytes are readily absorbed in both the small and large intestines, resulting in a fecal electrolyte content that is similar to that of plasma. Food and drink 2000 mL Saliva 1500 mL Gastric secretions 1500 mL Bile from liver 1000 mL Intestinal secretions 2000 mL (water and electrolytes) Feces 150 mL Pancreas 1000 mL Mucous secretions 200 mL Colon (up to 3000 mL of water reabsorbed) Figure 14–1 Fluid regulation in the gastrointestinal tract. 3920_Ch14_255-268 23/01/14 10:38 AM Page 257 Chapter 14 | Fecal Analysis The large intestine is capable of absorbing approximately 3000 mL of water. When the amount of water reaching the large intestine exceeds this amount, it is excreted with the solid fecal material, producing diarrhea. Constipation, on the other hand, provides time for additional water to be reabsorbed from the fecal material, producing small, hard stools. Diarrhea and Steatorrhea Diarrhea Diarrhea is defined as an increase in daily stool weight above 200 g, increased liquidity of stools, and frequency of more than three times per day. Diarrhea classification can be based on four factors: illness duration, mechanism, severity, and stool characteristics. Diarrhea lasting less than 4 weeks is defined as acute, and diarrhea persisting for more than 4 weeks is termed chronic diarrhea. The major mechanisms of diarrhea are secretory, osmotic, and intestinal hypermotility. The laboratory tests used to differentiate these mechanisms are fecal electrolytes (fecal sodium, fecal potassium), fecal osmolality, and stool pH. The normal total fecal osmolarity is close to the serum osmolality (290 mOsm/kg), normal fecal sodium is 30 mmol/L, and fecal potassium is 75 mmol/L. The fecal sodium and fecal potassium results are used to calculate the fecal osmotic gap. The fecal osmotic gap is calculated as follows: Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)] The osmotic gap in all forms of osmotic diarrhea is greater than 50 mOsm/kg and less than 50 mOsm/kg in secretory diarrhea. Electrolytes are increased in secretory diarrhea and negligible in osmotic diarrhea. A fecal fluid pH of less than 5.6 indicates a malabsorption of sugars, causing an osmotic diarrhea. disorders (hyperthyroidism, Zollinger-Ellison syndrome, VIPoma), neoplasms, and collagen vascular disease. Osmotic Diarrhea Osmotic diarrhea is caused by poor absorption that exerts osmotic pressure across the intestinal mucosa. Incomplete breakdown or reabsorption of food presents increased fecal material to the large intestine, resulting in water and electrolyte retention in the large intestine (osmotic diarrhea), which in turn results in excessive watery stool. Maldigestion (impaired food digestion) and malabsorption (impaired nutrient absorption by the intestine) contribute to osmotic diarrhea. The presence of unabsorbable solute increases the stool osmolality and the concentration of electrolytes is lower, resulting in an increased osmotic gap. Causes of osmotic diarrhea include disaccharidase deficiency (lactose intolerance), malabsorption (celiac sprue), poorly absorbed sugars (lactose, sorbitol, mannitol), laxatives, magnesium-containing antacids, amebiasis, and antibiotic administration. Laboratory testing of feces is frequently performed to aid in determining the cause of diarrhea (Table 14–1). Table 14–2 differentiates the features of osmotic diarrhea and secretory diarrhea. Table 14–1 Common Fecal Tests for Diarrhea Secretory Osmotic Stool cultures Ova and parasite examinations Rotavirus immunoassay Fecal leukocytes Microscopic fecal fats Muscle fiber detection Qualitative fecal fats Trypsin screening Microscopic fecal fats Muscle fiber detection Quantitative fecal fats Clinitest D-xylose tolerance test Lactose tolerance test Fecal electrolytes Stool pH Fecal osmolality Secretory Diarrhea Secretory diarrhea is caused by increased secretion of water. Bacterial, viral, and protozoan infections produce increased secretion of water and electrolytes, which override the reabsorptive ability of the large intestine, leading to secretory diarrhea. Enterotoxin-producing organisms such as Escherichia coli, Clostridium, Vibrio cholerae, Salmonella, Shigella, Staphylococcus, Campylobacter, protozoa, and parasites such as Cryptosporidium can stimulate these water and electrolyte secretions. Other causes of secretory diarrhea are drugs, stimulant laxatives, hormones, inflammatory bowel disease (Crohn disease, ulcerative colitis, lymphocytic colitis, diverticulitis), endocrine TECHNICAL TIP Process specimens for osmolality testing immediately. Specimens that are stored for hours may have a markedly increased osmolality due to the increased degradation of carbohydrates. 257 Table 14–2 Differential Features for Diarrhea Laboratory Test Osmotic Diarrhea Secretory Diarrhea Osmotic gap Stool Na Stool output in 24 hours pH Reducing substances >50 Osm/kg <60 mmol/L <200 g <50 Osm/kg >90 mmol/L >200 g <5.3 Positive >5.6 Negative 3920_Ch14_255-268 23/01/14 10:38 AM Page 258 258 Part Three | Other Body Fluids Altered Motility Altered motility describes conditions of enhanced motility (hypermotility) or slow motility (constipation). Both can be seen in irritable bowel syndrome (IBS), a functional disorder in which the nerves and muscles of the bowel are extra sensitive, causing cramping, bloating, flatus, diarrhea, and constipation. IBS can be triggered by food, chemicals, emotional stress, and exercise. Intestinal hypermotility is the excessive movement of intestinal contents through the GI tract that can cause diarrhea because normal absorption of intestinal contents and nutrients cannot occur. It can be caused by enteritis, the use of parasympathetic drugs, or with complications of malabsorption. Rapid gastric emptying (RGE) dumping syndrome describes hypermotility of the stomach and the shortened gastric emptying half-time, which causes the small intestine to fill too quickly with undigested food from the stomach. It is the hallmark of early dumping syndrome (EDS).1 Healthy people have a gastric emptying half-time range of 35 to 100 minutes, which varies with age and gender. A gastric emptying time of less than 35 minutes is considered RGE.1 RGE can be caused by disturbances in the gastric reservoir or in the transporting function. Alterations in the motor functions of the stomach result in accumulation of large amounts of osmotically active solids and liquids to be transported into the small intestine. Normal gastric emptying is controlled by fundic tone, duodenal feedback, and GI hormones. These are altered after gastric surgery, resulting in clinically significant dumping syndrome in approximately 10% of patients.2 RGE can be divided into early dumping and late dumping depending upon how soon after a meal the symptoms occur. EDS symptoms begin 10 to 30 minutes following meal ingestion.2 Symptoms include nausea, vomiting, bloating, cramping, diarrhea, dizziness, and fatigue. Late dumping occurs 2 to 3 hours after a meal and is characterized by weakness, sweating, and dizziness.1 Hypoglycemia is often a complication of dumping syndrome. The main causes of dumping syndrome include gastrectomy, gastric bypass surgery, post vagotomy status, Zollinger-Ellison syndrome, duodenal ulcer disease, and diabetes mellitus.1 Steatorrhea Detection of steatorrhea (fecal fat) is useful in diagnosing pancreatic insufficiency and small-bowel disorders that cause malabsorption. Absence of bile salts that assist pancreatic lipase in the breakdown and subsequent reabsorption of dietary fat (primarily triglycerides) produces an increase in stool fat (steatorrhea) that exceeds 6 g per day. Likewise, pancreatic disorders, including cystic fibrosis, chronic pancreatitis, and carcinoma, that decrease the production of pancreatic enzymes, are also associated with steatorrhea. Steatorrhea may be present in both maldigestion and malabsorption conditions and can be distinguished by the D-xylose test. D-Xylose is a sugar that does not need to be digested but does need to be absorbed to be present in the urine. If urine D-xylose is low, the resulting steatorrhea indicates a malabsorption condition. Malabsorption causes include bacterial overgrowth, intestinal resection, celiac disease, tropical sprue, lymphoma, Whipple disease, Giardia lamblia infestation, Crohn disease, and intestinal ischemia. A normal D-xylose test indicates pancreatitis. Specimen Collection Collection of a fecal specimen, frequently called a stool specimen, is not an easy task for patients. Detailed instructions and appropriate containers depending on the type of test and amount of feces required should be provided. For certain tests, dietary restrictions are required before fecal specimen collection. Patients should be instructed to collect the specimen in a clean container, such as a bedpan or disposable container, and transfer the specimen to the laboratory container. Patients should understand that the specimen must not be contaminated with urine or toilet water, which may contain chemical disinfectants or deodorizers that can interfere with chemical testing. Containers that contain preservatives for ova and parasites must not be used to collect specimens for other tests. Random specimens suitable for qualitative testing for blood and microscopic examination for leukocytes, muscle fibers, and fecal fats are usually collected in plastic or glass containers with screw-tops similar to those used for urine specimens. Material collected on a physician’s glove and samples applied to filter paper in occult blood testing kits are also received. For quantitative testing, such as for fecal fats, timed specimens are required. Because of the variability of bowel habits and the transit time required for food to pass through the digestive tract, the most representative sample is a 3-day collection. These specimens are frequently collected in large containers to accommodate the specimen quantity and facilitate emulsification before testing. Care must be taken when opening any fecal specimen to slowly release gas that has accumulated within the container. Patients must be cautioned not to contaminate the outside of the container. Macroscopic Screening The first indication of GI disturbances can often be changes in the brown color and formed consistency of the normal stool. Of course, the appearance of abnormal fecal color may also be caused by ingestion of highly pigmented foods and medications, so a differentiation must be made between this and a possible pathologic cause. Color The brown color of the feces results from intestinal oxidation of stercobilinogen to urobilin. As discussed in Chapter 5, conjugated bilirubin formed in the degradation of hemoglobin passes through the bile duct to the small intestine, where intestinal bacteria convert it to urobilinogen and stercobilinogen. Therefore, stools that appear pale (acholic stools) may signify a blockage of the bile duct. Pale stools are also associated with diagnostic procedures that use barium sulfate. 3920_Ch14_255-268 23/01/14 10:38 AM Page 259 Chapter 14 | Fecal Analysis A primary concern is the presence of blood in a stool specimen. Depending on the area of the intestinal tract from which bleeding occurs, the color can range from bright red to dark red to black. Blood that originates from the esophagus, stomach, or duodenum takes approximately 3 days to appear in the stool; during this time, degradation of hemoglobin produces the characteristic black, tarry stool. Likewise, blood from the lower GI tract requires less time to appear and retains its original red color. Both black and red stools should be chemically tested for the presence of blood, because ingestion of iron, charcoal, or bismuth often produces a black stool, and some medications and some foods, including beets, produce a red stool. Green stools may be observed in patients taking oral antibiotics, because of the oxidation of fecal bilirubin to biliverdin. Ingestion of increased amounts of green vegetables or food coloring also produces green stools. Appearance Besides variations in color, additional abnormalities may be observed during macroscopic examination. Table 14–3 lists common abnormalities seen in macroscopic evaluation. Examples include the watery consistency present in diarrhea; small, hard stools seen with constipation; and slender, ribbon-like stools, which suggest obstruction of the normal passage of material through the intestine. Table 14–3 Macroscopic Stool Characteristics12,26 Color/ Appearance Black Red Pale yellow, white, gray Green Bulky/frothy Ribbon-like Mucus- or blood-streaked mucus Possible Cause Upper GI bleeding Iron therapy Charcoal Bismuth (antacids) Lower GI bleeding Beets and food coloring Rifampin Bile-duct obstruction Barium sulfate Biliverdin/oral antibiotics Green vegetables Bile-duct obstruction Pancreatic disorders Intestinal constriction Colitis Dysentery Malignancy Constipation 259 Pale stools associated with biliary obstruction and steatorrhea appear bulky and frothy and frequently have a foul odor. Stools may appear greasy and may float. The presence of mucus-coated stools indicates intestinal inflammation or irritation. Mucus-coated stools may be caused by pathologic colitis, Crohn disease, colon tumors, or excessive straining during elimination. Blood-streaked mucus suggests damage to the intestinal walls, possibly caused by bacterial or amebic dysentery or malignancy. The presence of mucus should be reported. Microscopic Examination of Feces Microscopic screening of fecal smears is performed to detect the presence of leukocytes associated with microbial diarrhea and undigested muscle fibers and fats associated with steatorrhea. Fecal Leukocytes Leukocytes, primarily neutrophils, are seen in the feces in conditions that affect the intestinal mucosa, such as ulcerative colitis and bacterial dysentery. Microscopic screening is performed as a preliminary test to determine whether diarrhea is being caused by invasive bacterial pathogens including Salmonella, Shigella, Campylobacter, Yersinia, and enteroinvasive E. coli. Bacteria that cause diarrhea by toxin production, such as Staphylococcus aureus and Vibrio spp., viruses, and parasites usually do not cause the appearance of fecal leukocytes. Therefore, the presence or absence of fecal neutrophils can provide the physician with diagnostic information before receiving the culture report. Specimens can be examined as wet preparations stained with methylene blue or as dried smears stained with Wright’s or Gram stain. Methylene blue staining is the faster procedure but may be more difficult to interpret. Dried preparations stained with either Wright's or Gram stains provide permanent slides for evaluation. An additional advantage of the Gram stain is the observation of gram-positive and gram-negative bacteria, which could aid in the initial treatment.3 All slide preparations must be performed on fresh specimens. In an examination of preparations under high power, as few as three neutrophils per high-power field can be indicative of an invasive condition.4 Using oil immersion, the finding of any neutrophils has approximately 70% sensitivity for the presence of invasive bacteria.5 A lactoferrin latex agglutination test is available for detecting fecal leukocytes and remains sensitive in refrigerated and frozen specimens. The presence of lactoferrin, a component of granulocyte secondary granules, indicates an invasive bacterial pathogen.6 Muscle Fibers Microscopic examination of the feces for undigested striated muscle fibers can be helpful in diagnosing and monitoring 3920_Ch14_255-268 23/01/14 10:38 AM Page 260 260 Part Three | Other Body Fluids PROCEDURE 14-1 Methylene Blue Stain for Fecal Leukocytes 1. 2. 3. 4. 5. Place mucus or a drop of liquid stool on a slide. Add two drops of Löffler methylene blue. Mix with a wooden applicator stick. Allow to stand for 2 to 3 minutes. Examine for neutrophils under high power. patients with pancreatic insufficiency, such as in cases of cystic fibrosis. It is frequently ordered in conjunction with microscopic examinations for fecal fats. Increased amounts of striated fibers may also be seen in biliary obstruction and gastrocolic fistulas. Slides for muscle fiber detection are prepared by emulsifying a small amount of stool in 10% alcoholic eosin, which enhances the muscle fiber striations. The entire slide is examined for exactly 5 minutes, and the number of red-stained fibers with well-preserved striations is counted (Fig. 14–2). Care must be taken to correctly classify the fibers observed. Undigested fibers have visible striations running both vertically and horizontally. Partially digested fibers exhibit striations in only one direction, and digested fibers have no visible striations. Only undigested fibers are counted, and the presence of more than 10 is reported as increased (Fig. 14–3). To produce a representative sample, patients should be instructed to include red meat in their diet before collecting the specimen. Specimens should be examined within 24 hours of collection. Qualitative Fecal Fats Specimens from suspected cases of steatorrhea can be screened microscopically for the presence of excess fecal fat (steatorrhea). The procedure can also be used to monitor patients undergoing treatment for malabsorption disorders.7 In general, correlation Figure 14–2 Meat fibers present in fecal emulsion specimen using brightfield microscopy examination (×400). Figure 14–3 Note striations on meat fiber present in a fecal emulsion specimen (×1000). PROCEDURE 14-2 Muscle Fibers 1. Emulsify a small amount of stool in two drops of 10% eosin in alcohol. 2. Apply cover slip and let stand 3 minutes. 3. Examine under high power for 5 minutes. 4. Count the number of undigested fibers. between the qualitative and quantitative fecal fat procedures is good; however, additional unstained phospholipids and cholesterol esters are measured by the quantitative procedure.8,9 Lipids included in the microscopic examination of feces are neutral fats (triglycerides), fatty acid salts (soaps), fatty acids, and cholesterol. Their presence can be observed microscopically by staining with the dyes Sudan III, Sudan IV, or oil red O; Sudan III is the most routinely used. The staining procedure consists of two parts: the neutral fat stain and the split fat stain. Neutral fats are readily stained by Sudan III and appear as large orange-red droplets, often located near the edge of the cover slip.10 Observation of more than 60 droplets/high-power field can indicate steatorrhea; however, the split fat stain representing total fat content can provide a better indication.11 Breakdown of neutral fats by bacterial lipase and spontaneous hydrolysis of neutral fats may lower the neutral fat count, which precludes comparison of the two slide tests to determine whether maldigestion or malabsorption is causing steatorrhea. Soaps and fatty acids do not stain directly with Sudan III, so a second slide must be examined after the specimen has been mixed with acetic acid and heated. Examining this slide reveals stained droplets that represent not only the free fatty acids but also the fatty acids produced by hydrolysis of the soaps and the neutral fats (Fig. 14–4). In an examination of this split fat slide, both the number and size of the fat droplets must be considered. 3920_Ch14_255-268 23/01/14 10:39 AM Page 261 Chapter 14 | Fecal Analysis 261 Chemical Testing of Feces Occult Blood Figure 14–4 Several orange-red neutral fat globules present in a fecal suspension stained with Sudan III (×400). PROCEDURE 14-3 Neutral Fat Stain 1. Homogenize one part stool with two parts water. 2. Mix emulsified stool with one drop of 95% ethyl alcohol on slide. 3. Add two drops of saturated Sudan III in 95% ethanol. 4. Mix and apply cover slip. 5. Examine under high power. 6. Count orange droplets per high-power field. By far the most frequently performed fecal analysis is the detection of occult blood (hidden blood). As discussed previously, bleeding in the upper GI tract may produce a black, tarry stool, and bleeding in the lower GI tract may result in an overtly bloody stool. However, because any bleeding in excess of 2.5 mL/150 g of stool is considered pathologically significant, and no visible signs of bleeding may be present with this amount of blood, fecal occult blood testing (FOBT) is necessary. Annual testing for occult blood has a high positive predictive value for detecting colorectal cancer in the early stages and is recommended by the American Cancer Society, particularly for people older than age 50.13 Methods for detecting fecal occult blood include the guaiac, immunochemical, and fluorometric porphyrin quantification tests. Immunochemical tests and fecal porphyrin quantification tests are more sensitive and specific methods than the guaiac-based fecal occult blood tests. Guaiac-Based Fecal Occult Blood Tests The most frequently used screening test for fecal blood is the guaiac-based test for occult blood (gFOBT) based on detecting the pseudoperoxidase activity of hemoglobin. This is the same principle as the reagent strip test for urinary blood, but uses a different indicator chromogen. The reaction uses the pseudoperoxidase activity of hemoglobin reacting with hydrogen peroxide to oxidize a colorless compound to a colored compound. Pseudoperoxidase Normal specimens may contain as many as 100 small droplets, less than 4 µm in diameter, per high-power field. The same number of droplets measuring 1 to 8 µm is considered slightly increased, and 100 droplets measuring 6 to 75 µm is increased and commonly seen in steatorrhea.12 An increased amount of total fat on the second slide with normal fat content on the first slide is an indication of malabsorption, whereas maldigestion is indicated by increased neutral fat on the first slide. Cholesterol is stained by Sudan III after heating and as the specimen cools forms crystals that can be identified microscopically. PROCEDURE 14-4 Split Fat Stain 1. 2. 3. 4. 5. 6. Mix emulsified stool with one drop of 36% acetic acid. Add two drops of saturated Sudan III. Mix and apply cover slip. Heat gently almost to boiling. Examine under high power. Count and measure the orange droplets per highpower field. Hemoglobin + H2O2 + guaiac (colorless) oxidized guaiac + H2O (Blue color) Several different indicator chromogens have been used to detect occult blood. All react in the same chemical manner but vary in their sensitivity. Contrary to most chemical testing, the least sensitive reagent, guaiac, is preferred for routine testing. Considering that a normal stool can contain up to 2.5 mL of blood, a less sensitive chemical reactant is understandably more desirable. In addition, pseudoperoxidase activity is present from hemoglobin and myoglobin in ingested meat and fish, certain vegetables and fruits, and some intestinal bacteria. Therefore, to prevent false-positive reactions, test sensitivity must be decreased, which can be accomplished by varying the amount and purity of the guaiac reagent used in the test. Many commercial testing kits are available for occult blood testing with guaiac reagent. The kits contain guaiac-impregnated filter paper enclosed in a cardboard slide, to which the fecal specimen and hydrogen peroxide are added. Two or three filter paper areas are provided for application of material taken from different areas of the stool, and positive and negative controls are also included. Obtaining samples from the center of the stool avoids false-positive reactions from external contamination. The patient is asked to collect samples from stool specimens collected on 3 consecutive days. The sample is placed on the front side of the slide with an applicator stick and the 3920_Ch14_255-268 23/01/14 10:39 AM Page 262 262 Part Three | Other Body Fluids slide is closed. Adding hydrogen peroxide to the back of the filter paper slide that contains stool produces a blue color with guaiac reagent when pseudoperoxidase activity is present. Packaging of the guaiac-impregnated filter paper in individually sealed containers has facilitated colorectal cancer screening by allowing patients at home to place a specimen on a filter paper slide and bring or mail it to the laboratory for testing. To prevent false-positive reactions, specimens mailed to the laboratory should not be rehydrated before adding the hydrogen peroxide unless specifically instructed by the kit manufacturer. Specimens applied to the paper in the laboratory should be allowed to dry before testing. The specimens should be tested within 6 days of collection. Two samples from three different stools should be tested before a negative result is confirmed. To prevent the presence of dietary pseudoperoxidases in the stool, patients should be instructed to avoid eating red meats, horseradish, melons, raw broccoli, cauliflower, radishes, and turnips for 3 days before specimen collection. Aspirin and NSAIDs other than acetaminophen should not be taken for 7 days before specimen collection to prevent possible GI irritation. Vitamin C and iron supplements containing vitamin C should be avoided for 3 days before collections, because ascorbic acid is a strong reducing agent that interferes with the peroxidase reaction, causing a false-negative result.14 Immunochemical Fecal Occult Blood Test The immunochemical fecal occult blood test (iFOBT) is specific for the globin portion of human hemoglobin and uses polyclonal anti-human hemoglobin antibodies. Because this method is specific for human blood in feces, it does not require dietary or drug restrictions. It is more sensitive to lower GI bleeding that could be an indicator of colon cancer or other GI disease and can be used for patients who are taking aspirin and other anti-inflammatory medications. The iFOBT tests do not detect bleeding from other sources, such as a bleeding ulcer, thus decreasing the chance for false-positive reactions. Hemoglobin from upper GI bleeding is degraded by bacterial and digestive enzymes before reaching the large intestine and is immunochemically nonreactive. In contrast, there is little hemoglobin degradation in lower GI bleeding, so the blood is immunochemically active.15 Collection kits are similar to those used for guaiac testing, such as the Hemoccult ICT test (Beckman Coulter Inc., Fullerton, CA), and can be provided to patients for home collection. Depending on the method used, the results may be read visually or by an automated photometric instrument. Porphyrin-Based Fecal Occult Blood Test HemoQuant (SmithKline Diagnostics, Sunnyvale, CA) offers a porphyrin-based FOBT fluorometric test for hemoglobin based on the conversion of heme to fluorescent porphyrins. The test measures both intact hemoglobin and the hemoglobin that has been converted to porphyrins. As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that the gFOBT cannot detect, thereby making the HemoQuant test more sensitive to upper GI bleeding. False-negative results from upper GI bleeding can be seen with the gFOBT. Additionally, the porphyrin-based test is not affected by the presence of reducing or oxidizing substances or the water content of the fecal specimen. False-positive results can occur with the porphyrin-based test when non-human sources of blood (red meat) are present; therefore, patients should be instructed to avoid red meat for 3 days before the test. Quantitative Fecal Fat Testing Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea. As discussed, quantitative fecal analysis requires the collection of at least a 3-day specimen. The patient must maintain a regulated intake of fat (100 g/d) before and during the collection period. The specimen is collected in a large, preweighed container. Before analysis, the specimen is weighed and homogenized. Refrigerating the specimen prevents any bacterial degradation. The method routinely used for fecal fat measurement is the Van de Kamer titration, although gravimetric, near-infrared reflectance spectroscopy, and nuclear magnetic resonance spectroscopy methods are available.10 In the titration method, fecal lipids are converted to fatty acids and titrated to a neutral endpoint with sodium hydroxide. Approximately 80% of the total fat content is measured by titration, whereas the gravimetric method measures all fecal fat. A drawback of the titration/gravimetric method is that it is time consuming and uses corrosive and flammable solvents. A rapid (5 minutes) and safe procedure for analyzing quantitative fecal fat is the hydrogen nuclear magnetic resonance spectroscopy TECHNICAL TIP Failure to allow stool samples to soak into the filter paper slide for 3 to 5 minutes before adding developer may result in a false-negative result. SUMMARY 14-1 gFOBT Interference False-Positive Aspirin and anti-inflammatory medications Red meat Horseradish Raw broccoli, cauliflower, radishes, turnips Melons Menstrual and hemorrhoid contamination False-Negative Vitamin C >250 mg/d Iron supplements containing vitamin C Failure to wait specified time after sample is applied to add the developer reagent 3920_Ch14_255-268 23/01/14 10:39 AM Page 263 Chapter 14 | Fecal Analysis (1H NMR) method. In this method the homogenized specimen is microwaved-dried and analyzed. The results correlate well with the gravimetric method.16 The fat content is reported as grams of fat or the coefficient of fat retention per 24 hours. Reference values based on a 100 g/d intake are 1 to 6 g/d or a coefficient of fat retention of at least 95%. The coefficient of fat retention is calculated as follows: (dietary fat – fecal fat) × 100 (dietary fat) Although the Van de Kamer titration is the gold standard for fecal fat, the acid steatocrit is a rapid test to estimate the amount of fat excretion. It is similar to the microhematocrit test and is more convenient than a 72-hour stool collection. The acid steatocrit is a reliable tool to monitor a patient’s response to therapy and screen for steatorrhea in pediatric populations.17,18 PROCEDURE 14-5 Acid Steatocrit 1. Dilute 0.5 g of feces from a spot collection 1 to 4 with deionized water. 2. Vortex for 2 minutes to homogenize the specimen. 3. Add a volume of 5 N perchloric acid equal to 20% of the homogenate and then vortex the mixture for 30 seconds. Confirm the pH to be <1. 4. Place the acid-homogenate mixture in a 75-µL plain hematocrit capillary tube. Seal the end with wax. 5. Centrifuge the capillary tube horizontally at 13,000 rpm for 15 minutes in a microhematocrit centrifuge. This separates fat as an upper layer overlying a solid fecal layer. 6. Measure the length of the fat and solid layers using a magnifying lens. 7. Calculate the acid steatocrit in percent. 8. Calculate the fecal fat in grams per 24 hours. The acid steatocrit in percent = (fatty layer length in cm)/[(fatty layer length in cm) + (solid layer length)] × 100 The fecal fat for adults is quantitated as follows: Fecal fat in grams per 24 hours = [0.45 × (acid steatocrit in percent as a whole number)] – 0.43 An acid steatocrit value <31% is considered normal while a value >31% indicates steatorrhea in adults. The fecal fat for children up to the age of 15 years is as follows: Fecal fat in grams per 24 hours = [0.1939 × (acid steatocrit in percent as a whole number)] – 0.2174 Acid steatocrit is higher in infants and lowers with age.19 An acid steatocrit of <10% indicates steatorrhea in children.18 263 Near-infrared reflectance spectroscopy (NIRS) is a rapid procedure for fecal fat that requires less stool handling by laboratory personnel. The test requires a 48- to 72-hour stool collection to exclude day-to-day variability, but it does not require reagents after homogenization of the sample. The result is based on the measurement and computed processing of signal data from reflectance of fecal surface, which is scanned with infrared light between 1400 nM and 2600 nM wavelength. The results are calculated from calibration derived from known samples. The technique quantitates water, fat, and nitrogen in grams per 24 hours.20 A summary of tests and current instrumentation for fecal fat analysis is presented in Table 14–4. APT Test (Fetal Hemoglobin) Grossly bloody stools and vomitus are sometimes seen in neonates as the result of swallowing maternal blood during delivery. Should it be necessary to distinguish between the presence of fetal blood or maternal blood in an infant’s stool or vomitus, the APT test may be requested. The material to be tested is emulsified in water to release hemoglobin (Hb) and, after centrifugation, 1% sodium hydroxide is added to the pink hemoglobin-containing supernatant. In the presence of alkali-resistant fetal hemoglobin, the solution remains pink (HbF), whereas denaturation of the maternal hemoglobin (HbA) produces a yellow-brown supernatant after standing for 2 minutes. The APT test distinguishes not only between HbA and HbF but also between maternal hemoglobins AS, CS, and SS and HbF. The presence of maternal thalassemia major would produce erroneous results owing to the high concentration of HbF. Stool specimens should be tested when fresh. They may appear bloody but should not be black and tarry, because this would indicate already denatured hemoglobin.21 Table 14–4 Tests, Materials, and Instrumentation for Fecal Fat Analysis19 Procedure Materials, Instrumentation Sudan III Steatocrit and acid steatocrit Fecal elastase–I Near-infrared reflectance spectroscopy (NIRS) Van de Kamer titration Sudan stain, microscopy Hematocrit centrifuge, gravimetric assay Immunoassay ELISA technique NIRS spectrophotometer with specialized computer software Nuclear magnetic resonance spectroscopy Fecal fat extraction and titration of long chain fatty acid by sodium hydroxide Microwaved-dried specimen; hydrogen nuclear magnetic spectrophotometer 3920_Ch14_255-268 23/01/14 10:39 AM Page 264 264 Part Three | Other Body Fluids PROCEDURE 14-6 APT Test 1. 2. 3. 4. 5. 6. 7. Emulsify specimen in water. Centrifuge. Divide pink supernatant into two tubes. Add 1% sodium hydroxide to one tube. Wait 2 minutes. Compare color with that in the control tube. Prepare controls using cord blood and adult blood. Fecal Enzymes Enzymes supplied to the gastrointestinal tract by the pancreas are essential for digesting dietary proteins, carbohydrates, and fats. Decreased production of these enzymes (pancreatic insufficiency) is associated with disorders such as chronic pancreatitis and cystic fibrosis. Steatorrhea occurs, and undigested food appears in the feces. Analysis of the feces focuses primarily on the proteolytic enzymes trypsin, chymotrypsin, and elastase I. Fecal chymotrypsin is more resistant to intestinal degradation and is a more sensitive indicator of less severe cases of pancreatic insufficiency. It also remains stable in fecal specimens for up to 10 days at room temperature. Chymotrypsin is capable of gelatin hydrolysis but is most frequently measured by spectrophotometric methods. Elastase I is an isoenzyme of the enzyme elastase and is the enzyme form produced by the pancreas. It is present in high concentrations in pancreatic secretions and is strongly resistant to degradation. It accounts for about 6% of all secreted pancreatic enzymes.22 Fecal elastase I is pancreas specific and its concentration is about five times higher than in pancreatic juice. It is not affected by motility disorders or mucosal defects.23 Elastase I can be measured by immunoassay using HISTORICAL NOTE Screening Test for Fecal Trypsin Historically, absence of trypsin has been screened for by exposing x-ray paper to stool emulsified in water. When trypsin is present in the stool, it digests the gelatin on the paper, leaving a clear area. Inability to digest the gelatin indicates a deficiency in trypsin production. The gelatin test is an insensitive procedure that detects only severe cases of pancreatic insufficiency. In addition, false-negative results may occur as the result of intestinal degradation of trypsin and the possible presence of trypsin inhibitors in the feces. The proteolytic activity of bacteria enzymes may produce false-positive results in old specimens. the ELISA kit and provides a very sensitive indicator of exocrine pancreatic insufficiency.24,25 It is easy to perform and requires only a single stool sample. The ELISA test uses monoclonal antibodies against human pancreatic elastase-1; therefore, the result is specific for human enzyme and not affected by pancreatic enzyme replacement therapy.22 The test is specific in differentiating pancreatic from nonpancreatic causes in patients with steatorrhea.23 Carbohydrates The presence of increased carbohydrates in the stool produces osmotic diarrhea from the osmotic pressure of the unabsorbed sugar in the intestine drawing in fluid and electrolytes. Carbohydrates in the feces may be present as a result of intestinal inability to reabsorb carbohydrates, as is seen in celiac disease, or lack of digestive enzymes such as lactase resulting in lactose intolerance. Idiopathic lactase deficiency is common, predominantly occurring in the African, Asian, and southern European Greek populations. Carbohydrate malabsorption or intolerance (maldigestion) is primarily analyzed by serum and urine tests; however, an increased concentration of carbohydrate can be detected by performing a copper reduction test on the fecal specimen. Testing for fecal reducing substances detects congenital disaccharidase deficiencies as well as enzyme deficiencies due to nonspecific mucosal injury. Fecal carbohydrate testing is most valuable in assessing cases of infant diarrhea and may be accompanied by a pH determination. Normal stool pH is between 7 and 8; however, increased use of carbohydrates by intestinal bacterial fermentation increases the lactic acid level and lowers the pH to below 5.5 in cases of carbohydrate disorders. The copper reduction test is performed using a Clinitest tablet (Siemens Healthcare Diagnostics, Inc., Deerfield, IL) and one part stool emulsified in two parts water. A result of 0.5 g/dL is considered indicative of carbohydrate intolerance. The Clinitest on stools can distinguish between diarrhea caused by abnormal excretion of reducing sugars and those caused by various viruses and parasites. Sucrose is not detected by the Clinitest method because it is not a reducing sugar. In premature infants there is correlation between a positive Clinitest and inflammatory necrotizing enterocolitis. As discussed in Chapter 5, this is a general test for the presence of reducing substances, and a positive result would be followed by more specific serum carbohydrate tolerance tests, the most common being the D-xylose test for malabsorption and the lactose tolerance test for maldigestion. Stool chromatography to identify the malabsorbed carbohydrate is available but rarely necessary to diagnose sugar intolerance. Small-bowel biopsy specimens for histologic examination and the assay of disaccharidase enzyme activity differentiate primary from secondary disaccharidase intolerance.26 A summary of fecal screening tests is presented in Table 14–5. 3920_Ch14_255-268 23/01/14 10:39 AM Page 265 Chapter 14 | Fecal Analysis Table 14–5 265 Fecal Screening Tests Test Methodology/Principle Interpretation Examination for neutrophils Qualitative fecal fats Microscopic count of neutrophils in smear stained with methylene blue, Gram stain, or Wright's stain Microscopic examination of direct smear stained with Sudan III Microscopic examination of smear heated with acetic acid and Sudan III Pseudoperoxidase activity of hemoglobin liberates oxygen from hydrogen peroxide to oxidize guaiac reagent Uses polyclonal anti-human antibodies specific for the globin portion of human hemoglobin Addition of sodium hydroxide to hemoglobin-containing emulsion determines presence of maternal or fetal blood Emulsified specimen placed on x-ray paper determines ability to digest gelatin Immunoassay using an ELISA test Three per high-power field indicates condition affecting intestinal wall 60 large orange-red droplets indicates malabsorption 100 orange-red droplets measuring 6 to 75 µm indicates malabsorption Blue color indicates gastrointestinal bleeding Positive test and control lines indicate GI bleeding Pink color indicates presence of fetal blood gFOBT iFOBT APT test Trypsin Elastase 1 Clinitest Addition of Clinitest tablet to emulsified stool detects presence of reducing substances Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Singh, A, Gull, H, and Singh, R: Clinical significance of rapid (accelerated) gastric emptying. Clin Nucl Med 28(8):652–658, 2003. 2. Ukleja, A: Dumping syndrome: Pathophysiology and treatment. Nutr Clin Pract 20(5):517–525, 2005. 3. Koepke, JA: Tips from the clinical experts. MLO, p. 15, 1995. 4. Bradley, GM: Fecal analysis: Much more than an unpleasant necessity. Diagn Med 3(2):64–75, 1980. 5. Novak, R, et al: How useful are fecal neutrophil determinations? Lab Med 26(11):433, 1995. 6. McCray, WH, and Krevsky, B: Diagnosing diarrhea in adults: A practical approach. Hosp Med 34(4):27–36, 1998. 7. Walters, MP, et al: Clinical monitoring of steatorrhea in cystic fibrosis. Arch Dis Child 65:99–102, 1990. 8. Khouri, MR, Huang, G, and Shiau, YF: Sudan stain of fecal fat: New insight into an old test. Gastroenterology 96(2 Pt 1): 421–427, 1990. 9. Simko, V: Fecal fat microscopy. Am J Gastroenterol 75(3): 204–208, 1981. 10. Van de Kamer, JH, et al: A rapid method for determination of fat in feces. J Biol Chem 177:347–355, 1949. 11. Freeman, JA, and Beeler, MF: Laboratory Medicine: Urinalysis and Medical Microscopy. Lea & Febiger, Philadelphia, 1983. 12. Drummey, GD, Benson, JA, and Jones, CM: Microscopic examination of the stool for steatorrhea. N Engl J Med 264:85–87, 1961. 13. Mandel, JS, et al: The effect of fecal occult-blood screening on the incidence of colorectal cancer. N Engl J Med 343(22): 1603–1607, 2000. Inability to digest gelatin indicates lack of trypsin Sensitive indicator of exocrine pancreatic insufficiency Reaction of 0.5 g/dL reducing substances suggests carbohydrate intolerance 14. Knight, KK, Fielding, JE, and Battista, RN: Occult blood screening for colorectal cancer. JAMA 261:587–590, 1989. 15. Hemoccult ICT immunochemical fecal occult blood test package insert. Beckman Coulter, Fullerton, CA, May 2005. 16. Korpi-Steiner, N, Ward, JN, Kumar, V, and McConnell, JP: Comparative analysis of fecal fat quantitation via nuclear magnetic resonance spectroscopy (1H NMR and gravimetry. Clin Chim Acta 400:33, 2009. 17. Bijoor, AR, Geetha, S, and Venkateash, T: Faecal fat content in healthy adults by the “acid steatocrit method.” Indian J Clin Biochem 19(2):20–22, 2004. 18. Van den Neucker, AM, et al: Acid steatocrit: a reliable screening tool for steatorrhoea. Acta Paediatr 90:873–875, 2001. 19. Guarino, A, et al: Reference values of the steatocrit and its modifications in diarrheal diseases. J Pediatr Gastroenterol Nutr 14:268–274, 1992. 20. Serrano, PL, Navarro, JLL, and Fernandez-Rodriguez, CM: Laboratory tests and equipment for diagnostic work-up for malabsorption syndrome. LABTECH 2004. 21. Croak, M: Haemoglobin in stools from neonates: Measurement by a modified APT test. Med Lab Sci 48(4):346–350, 1991. 22. Elphick, DA, and Kapur, K: Comparing the urinary pancreolauryl ratio and faecal elastase-1 as indicators of pancreatic insufficiency in clinical practice. Pancreatology 5:196–200, 2005. 23. Symersky, T, et al: Faecal elastase-I: Helpful in analysing steatorrhoea? Neth J Med 62(8):286–289, 2004. 24. Phillips, IJ, et al: Faecal elastase I: A marker of exocrine pancreatic insufficiency in cystic fibrosis. Ann Clin Chem 36:739–742, 1999. 25. Thorne, D, and O’Brien, C: Diagnosing chronic pancreatitis. Advance 12(14):8–12, 2000. 26. Robayo-Torres, CC, Quezada-Calvillo, R, and Nichols, BL: Disaccharide digestion: Clinical and molecular aspects. Clin Gastroenterol Hepatol 4(3):276–287, 2006. 3920_Ch14_255-268 23/01/14 10:39 AM Page 266 266 Part Three | Other Body Fluids Study Questions 1. In what part of the digestive tract do pancreatic enzymes and bile salts contribute to digestion? 8. Stools from persons with steatorrhea will contain excess amounts of: A. Large intestine A. Barium sulfate B. Liver B. Blood C. Small intestine C. Fat D. Stomach D. Mucus 2. Where does the reabsorption of water take place in the primary digestive process? A. Large intestine B. Pancreas C. Small intestine D. Stomach 3. Which of the following tests is not performed to detect osmotic diarrhea? A. Clinitest B. Fecal fats C. Fecal neutrophils D. Muscle fibers 4. The normal composition of feces includes all of the following except: 9. Which of the following pairings of stool appearance and cause does not match? A. Black, tarry: blood B. Pale, frothy: steatorrhea C. Yellow-gray: bile duct obstruction D. Yellow-green: barium sulfate 10. Stool specimens that appear ribbon-like are indicative of which condition? A. Bile-duct obstruction B. Colitis C. Intestinal constriction D. Malignancy 11. A black tarry stool is indicative of: A. Bacteria A. Upper GI bleeding B. Blood B. Lower GI bleeding C. Electrolytes C. Excess fat D. Water D. Excess carbohydrates 5. What is the fecal test that requires a 3-day specimen? A. Fecal occult blood 12. Chemical screening tests performed on feces include all of the following except: B. APT test A. APT test C. Elastase I B. Clinitest D. Quantitative fecal fat testing C. Pilocarpine iontophoresis 6. The normal brown color of the feces is produced by: A. Cellulose D. Quantitative fecal fats 13. Secretory diarrhea is caused by: B. Pancreatic enzymes A. Antibiotic administration C. Undigested foodstuffs B. Lactose intolerance D. Urobilin 7. Diarrhea can result from all of the following except: A. Addition of pathogenic organisms to the normal intestinal flora C. Celiac sprue D. Vibrio cholerae 14. The fecal osmotic gap is elevated in which disorder? B. Disruption of the normal intestinal bacterial flora A. Dumping syndrome C. Increased concentration of fecal electrolytes B. Osmotic diarrhea D. Increased reabsorption of intestinal water and electrolytes C. Secretory diarrhea D. Steatorrhea 3920_Ch14_255-268 23/01/14 10:39 AM Page 267 Chapter 14 | Fecal Analysis 15. Microscopic examination of stools provides preliminary information as to the cause of diarrhea because: A. Neutrophils are present in conditions caused by toxin-producing bacteria B. Neutrophils are present in conditions that affect the intestinal wall C. Red and white blood cells are present if the cause is bacterial D. Neutrophils are present if the condition is of nonbacterial etiology 16. True or False: The presence of fecal neutrophils would be expected with diarrhea caused by a rotavirus. 17. Large orange-red droplets seen on direct microscopic examination of stools mixed with Sudan III represent: A. Cholesterol B. Fatty acids C. Neutral fats D. Soaps 18. Microscopic examination of stools mixed with Sudan III and glacial acetic acid and then heated will show small orange-red droplets that represent: A. Fatty acids and soaps B. Fatty acids and neutral fats C. Fatty acids, soaps, and neutral fats D. Soaps 19. When performing a microscopic stool examination for muscle fibers, the structures that should be counted: A. Are coiled and stain blue B. Contain no visible striations C. Have two-dimensional striations D. Have vertical striations and stain red 20. A value of 85% fat retention would indicate: A. Dumping syndrome B. Osmotic diarrhea C. Secretory diarrhea D. Steatorrhea 21. Which of the following tests would not be indicative of steatorrhea? A. Fecal elastase-I B. Fecal occult blood C. Sudan III D. Van de Kamer 22. The term “occult” blood describes blood that: A. Is produced in the lower GI tract B. Is produced in the upper GI tract C. Is not visibly apparent in the stool specimen D. Produces a black, tarry stool 267 23. What is the recommended number of samples that should be tested to confirm a negative occult blood result? A. One random specimen B. Two samples taken from different parts of three stools C. Three samples taken from the outermost portion of the stool D. Three samples taken from different parts of two stools 24. The immunochemical tests for occult blood: A. Test for human globulin B. Give false-positive reactions with meat hemoglobin C. Can give false-positive reactions with aspirin D. Are inhibited by porphyrin 25. Guaiac tests for detecting occult blood rely on the: A. Reaction of hemoglobin with hydrogen peroxide B. Pseudoperoxidase activity of hemoglobin C. Reaction of hemoglobin with ortho-toluidine D. Pseudoperoxidase activity of hydrogen peroxide 26. What is the significance of an APT test that remains pink after addition of sodium hydroxide? A. Fecal fat is present. B. Fetal hemoglobin is present. C. Fecal trypsin is present. D. Vitamin C is present. 27. In the Van de Kamer method for quantitative fecal fat determinations, fecal lipids are: A. Converted to fatty acids prior to titrating with sodium hydroxide B. Homogenized and titrated to a neutral endpoint with sodium hydroxide C. Measured gravimetrically after washing D. Measured by spectrophotometer after addition of Sudan III 28. A patient whose stool exhibits increased fats, undigested muscle fibers, and the inability to digest gelatin may have: A. Bacterial dysentery B. A duodenal ulcer C. Cystic fibrosis D. Lactose intolerance 3920_Ch14_255-268 23/01/14 10:39 AM Page 268 268 Part Three | Other Body Fluids 29. A stool specimen collected from an infant with diarrhea has a pH of 5.0. This result correlates with a: A. Positive APT test B. Negative trypsin test C. Positive Clinitest D. Negative occult blood test 30. Which of the following tests differentiates a malabsorption cause from a maldigestion cause in steatorrhea? A. APT test B. D-xylose test C. Lactose tolerance test D. Occult blood test Case Studies and Clinical Situations 1. Microscopic screening of a stool from a patient exhibiting prolonged diarrhea shows increased fecal neutrophils and normal qualitative fecal fats and meat fibers. a. What type of diarrhea do these results suggest? b. Name an additional test that could provide more diagnostic information. c. Name one probable result for this test and one improbable result. d. If the test for fecal neutrophils were negative and the fecal fat concentration increased, what type of diarrhea would be suggested? 2. Laboratory studies are being performed on a 5-year-old boy to determine whether there is a metabolic reason for his continued failure to gain weight. In addition to having blood drawn, the patient has a sweat chloride collected, provides a random stool sample, and is asked to collect a 72-hour stool sample. a. How can the presence of steatorrhea be screened for by testing the random stool sample? b. How does this test distinguish among neutral fats, soaps, and fatty acids? c. What confirmatory test should be performed? d. Describe the appearance of the stool specimens if steatorrhea is present. e. If a diagnosis of cystic fibrosis is suspected, state two screening tests that could be performed on a stool specimen to aid in the diagnosis. f. State a possible reason for a false-negative reaction in each of these tests. g. What confirmatory test could be performed? 3. A physician’s office laboratory is experiencing inconsistencies in the results of patient-collected specimens for FOBT. Patients are instructed to submit samples from two areas of three different stools. Positive and negative controls are producing satisfactory results. Patient #1 is a 30year-old woman taking over-the-counter medications for gastric reflux who has reported passing frequent, black stools. The results of all three specimens are negative for occult blood. Patient #2 is a 70-year-old woman suffering from arthritis. She is taking the test as part of a routine physical. The results of all three specimens are positive for occult blood. Patient #3 is a 50-year-old man advised by the doctor to lose 30 lb. He has been doing well on a high-protein, low-carbohydrate diet. Two of his three specimens are positive for occult blood. a. What is the possible nonpathologic cause of the unexpected results for Patient #1? Patient #2? Patient #3? b. How could the physician’s office staff avoid these discrepancies? c. What testing methodology could be used for Patients #2 and #3? 4. A watery black stool from a neonate is received in the laboratory with requests for an APT test, fecal pH, and a Clinitest. a. Can all three tests be performed on this specimen? Why? b. If the Clinitest is positive, what pH reading can be expected? Why? c. The infant’s hemoglobin remains constant at 18 g/dL. What was the significance of the black stool? d. Would this infant be expected to have ketonuria? Why or why not? 3920_Ch15_269-282 23/01/14 10:40 AM Page 269 CHAPTER 15 Vaginal Secretions LEARNING OBJECTIVES Upon completing this chapter, the reader will be able to: 15-1 State the indications for collecting vaginal specimens. 15-2 Describe the specimen collection and handling procedures for vaginal specimens and explain how deviations from the correct practice will affect test results. 15-3 Describe the appearance of normal and abnormal vaginal secretions. 15-4 Explain the significance of vaginal pH values. 15-5 List the diagnostic tests performed on vaginal secretions and explain the appropriate use for each. 15-6 Describe the microscopic constituents for the common syndromes associated with vaginitis. 15-7 Identify the most common causes of vaginitis including the cause, clinical signs and symptoms, laboratory tests, and treatment. 15-8 Describe two tests that can be performed on vaginal secretions to predict conditions of premature delivery and rupture of fetal membranes. KEY TERMS Atrophic vaginitis Dysuria Trichomonas vaginalis Bacterial vaginosis (BV) Gardnerella vaginalis Trichomoniasis Basal cells Lactobacilli Vaginal pool Clue cells Mobiluncus spp. Vaginitis Desquamative inflammatory vaginitis (DIV) Parabasal cells Vulvovaginal candidiasis Pruritus Yeast Dyspareunia 3920_Ch15_269-282 23/01/14 10:40 AM Page 270 270 Part Three | Other Body Fluids Vaginal secretions are examined in the clinical laboratory to diagnose infections and complications of pregnancy, and for forensic testing (see Chapter 10, Semen) in sexual assault patients. In this chapter, the most commonly encountered disorders and the diagnostic laboratory tests used to evaluate vaginal secretions will be discussed. Vaginitis is one of the most common conditions diagnosed by health-care providers for female patients, particularly women of childbearing age. It is characterized by abnormal vaginal discharge or odor, pruritus, vaginal irritation, dysuria, and dyspareunia. Most often, vaginitis is secondary to bacterial vaginosis (BV), vulvovaginal candidiasis, or trichomoniasis; however, vaginitis can also occur with non-infectious conditions such as vaginal atrophy, allergies, and chemical irritation.1 Although the symptoms for the various syndromes of vaginitis are similar, the effective treatment for each depends on an accurate diagnosis. Careful microscopic examination of fresh vaginal secretions is necessary to determine the causative agent for each syndrome and to provide the appropriate treatment for the patient and, in some cases, the sexual partners, to avoid reinfection. Microscopic methods include a saline wet mount examination, potassium hydroxide (KOH) examination, and the Gram stain, which is considered the gold standard. Clinical laboratory personnel performing urine microscopic examinations should be aware that microscopic constituents observed in vaginal fluid may also be seen in urine specimens when the urine specimen is contaminated with vaginal secretions. Other tests used for differential diagnosis include litmus pH levels, DNA probe testing, culture, and point of care test kits. The clinical and microscopic features of the common syndromes are summarized in Table 15–1. Table 15–1 Findings Appearance pH WBCs Lactobacilli Clue cells Other cells In addition to evaluating vaginal secretions for infections, tests are performed on vaginal secretions to detect the placental ␣ 1-microglobulin (PAMG-1) protein to diagnose ruptured fetal membranes, or fetal fibronectin enzyme to assess the risk of preterm delivery. The fern test (see Chapter 13) is used to identify amniotic fluid that may be present when the amniotic sac has ruptured.2 Specimen Collection and Handling The health-care provider collects vaginal secretions during a pelvic examination. Detailed instructions and the specific manufacturer’s collection and transport devices must be provided and are specific to the organism sought. Correct specimen handling and timely transport to the laboratory is important for optimal detection of the responsible pathogen. A speculum moistened with warm water is used to visualize the vaginal fornices. Lubricants may contain antibacterial agents and must not be used. The specimen is collected by swabbing the vaginal walls and vaginal pool to collect epithelial cells along with the vaginal secretions using one or more sterile, polyester-tipped swabs on a plastic shaft or swabs specifically designated by the manufacturer.3 Cotton swabs should not be used because cotton is toxic to Neisseria gonorrhoeae, the wood in a wooden shaft may be toxic to Chlamydia trachomatis, and calcium alginate can inactivate herpes simplex virus (HSV) for viral cultures.4 The health-care provider performs a gross examination of the vaginal secretions and then places the swab in a tube containing 0.5 to 1.0 mL of sterile physiologic saline. The tube is Clinical Features and Laboratory Findings in Vaginitis2 Bacterial Vaginosis Candidiasis Trichomoniasis Thin, homogeneous, whiteto-gray vaginal discharge >4.5 Rare or absent Rare or absent White, curd-like vaginal discharge Yellow-green frothy adherent vaginal discharge increased in volume >4.5 2+ to 4+ Absent or present >20% Absent Large clumps of epithelial cells 3.8 to 4.5 3+ to 4+ Present Desquamative Inflammatory Vaginitis Atrophic Vaginitis Excessive purulent vaginal discharge, vaginal erythema >4.5 3+ to 4+ Absent or reduced Excessive purulent vaginal discharge, vaginal erythema >4.5 3+ to 4+ Decreased Occasional parabasal or basal cells >1+ RBCs Occasional parabasal or basal cells >1+ RBCs Absent or present 3920_Ch15_269-282 23/01/14 10:40 AM Page 271 Chapter 15 | Vaginal Secretions Table 15–1 Findings 271 Clinical Features and Laboratory Findings in Vaginitis2—cont’d Bacterial Vaginosis Candidiasis Trichomoniasis Desquamative Inflammatory Vaginitis Other organisms Increase in small curved bacilli, coccobacilli, and pleomorphic bacilli Budding yeast cells and pseudohyphae Trichomonas frequently associated with other organisms 2+ grampositive cocci Amine (Whiff) test Other tests Positive Negative Positive Negative Confirmatory tests: DNA probe, proline amino peptidase, OSOM BVBLUE Rapid Test Confirmatory tests: DNA probe, OSOM BVBLUE Rapid Test Confirmatory tests: DNA probe or culture, OSOM Trichomonas Rapid Test sealed for transport to the laboratory, where the specimen is processed for microscopic analysis. The swab should be vigorously twirled in the saline to dislodge particulates from the swab. Failure to dislodge particles may lead to erroneous results. Specimens should be tested with pH paper before being placed in saline.2 An alternative method of specimen preparation is to dilute a sample of vaginal discharge in one to two drops of normal saline solution directly on a microscope slide. A second sample is then placed in 10% KOH solution in the same manner. Cover slips are placed over both slides for microscopic examination.5 Properly labeled specimens should be placed in a biohazard bag with the requisition and transported to the laboratory as soon as possible. The requisition must include the patient’s name and unique identifier as well as a patient medical history that should include menstrual status; use of vaginal creams, lubricants, and douches; and recent exposure to sexually transmitted diseases.3 Specimens should be analyzed immediately, but if a delay in transport or analysis is necessary, specimen handling is based on the suspected pathogen. Specimens must be kept at room temperature to preserve the motility of Trichomonas vaginalis and the recovery of N. gonorrhoeae, whereas specimens for C. trachomatis and herpes simplex virus must be refrigerated to prevent overgrowth of normal flora.4 Specimens for T. vaginalis should be examined within 2 hours of collection.2 Color and Appearance Normal vaginal fluid appears white with a flocculent discharge. Microscopically, normal vaginal flora includes a predominance of large, rod-shaped, gram-positive lactobacilli and squamous Atrophic Vaginitis Increased grampositive cocci and gramnegative rods; decreased large rods Negative epithelial cells. White blood cells may be present and red blood cells will be present if the patient is menstruating3 (Table 15–2). Abnormal vaginal secretions may appear as an increased thin, homogeneous white-to-gray discharge often seen in bacterial vaginosis, or as a white “cottage cheese”–like discharge particular for Candida infections, or as an increased yellow-green, frothy, adherent discharge associated with T. vaginalis.3 C. trachomatis may present with a yellow, opaque cervical discharge.6 Diagnostic Tests pH The health-care provider can perform a vaginal pH test when performing a pelvic examination. The test should be performed before placing the swab into saline or KOH solutions. Table 15–2 Normal Findings in Vaginal Secretions Appearance pH Amine (Whiff) test WBCs Lactobacilli Clue cells Other cells Other organisms White, flocculent discharge 3.8 to 4.2 Negative 2+ Predominant Absent Absent (except RBCs during menses) Other lactobacilli subgroups, occasional yeast 3920_Ch15_269-282 23/01/14 10:40 AM Page 272 272 Part Three | Other Body Fluids Commercial pH test paper with a narrow pH range is recommended to more accurately evaluate pH values in the 4.5 range. The test paper is placed in the pooled vaginal secretion and the color change is compared to a chart with corresponding pH values. Factors that can interfere with the pH test include contamination of the vaginal secretions with cervical mucus, semen, and blood.6 The pH test helps to differentiate the causes of vaginitis, as shown in Table 15–1. The vaginal pH is usually about 4.5 in women with vulvovaginal candidiasis but is above 4.5 in women with bacterial vaginosis, trichomoniasis, desquamative inflammatory vaginitis (DIV), and atrophic vaginitis.1,2,4 As described previously, normal vaginal flora includes a predominance of the bacteria lactobacilli, which produce the end product lactic acid from glycogen metabolism. Lactic acid provides an acidic vaginal environment with a pH value between 3.8 and 4.5. This acidity suppresses the overgrowth of infectious organisms such as Mobiluncus, Prevotella, and Gardnerella vaginalis, and therefore maintains the balance of normal vaginal bacteria flora.7 Some lactobacilli subgroups also produce hydrogen peroxide, which is toxic to pathogens, and helps keep the vaginal pH acidic to provide protection from urogenital infections. Bacterial vaginosis has been associated with the absence of hydrogen peroxide–producing lactobacilli.3 Estrogen production also is necessary to preserve an acidic vaginal environment.6 Microscopic Procedures Vaginal infections are usually diagnosed from microscopic examination. Saline wet mounts and KOH mounts are the initial screening tests and the Gram stain is used as a confirmatory examination for yeast or bacterial vaginosis.2 Slides are prepared from the saline specimen solution that was made from the vaginal swab immediately after collection. Three clean glass slides (if a Gram stain is requested) are labeled with the patient’s name and a unique identifier. A drop of specimen is placed on each slide using a disposable transfer pipette. An alternative method is to press the swab against the slide and then roll the swab over the slide. The slide for Gram stain is allowed to dry and then heat-fixed for the Gram stain procedure and examination performed in the microbiology section of the laboratory. For wet mount examinations, cells and organisms are quantified per high power field (hpf) (40×); for Gram stains, cells and organisms are reported per oil immersion field (100×). Wet Mount Examination For the saline wet mount examination, a cover slip is placed on the specimen carefully to exclude air bubbles. The slide is examined microscopically using the low power (10×) and high dry power (40×) objective with a bright-field microscope. Using the low power objective (100×magnification), the slide is scanned for an even distribution of cellular components, types and numbers of epithelial cells, clumping of epithelial cells, and the presence of budding yeast or pseudohyphae. The slide is then examined using the high power objective (400× magnification) and the organisms and cells are counted and reported per high power field (hpf) using the criteria in Table 15–3. Typical constituents found in vaginal fluid wet mounts include squamous epithelial cells, white blood cells, red blood cells, clue cells, parabasal cells, basal cells, bacteria, motile Trichomonas vaginalis, yeast, and hyphae/pseudohyphae. Intravaginal medications might leave oil droplets that can interfere with the interpretation of wet mounts. In this case, a Gram stain is useful to detect yeast or bacterial vaginosis.2 Squamous Epithelial Cells Squamous epithelial cells measure 25 to 70 µm in diameter and exhibit a polygonal “flagstone” appearance. They contain a prominent centrally located nucleus about the size of a red blood cell and a large amount of irregular cytoplasm, lacking granularity, with distinct cell margins. (Fig. 15–1) These large, flat cells originate from the linings of the vagina and female urethra and are present in significant numbers in the vaginal Table 15–3 Rare 1+ 2+ 3+ 4+ Quantitation Scheme for Microscopic Examinations 2 Less than 10 organisms or cells/slide Less than 1 organism or cell/hpf 1 to 5 organisms or cells/hpf 6 to 30 organisms or cells/hpf >30 organisms or cells/hpf PROCEDURE 15-1 pH Test 1. Using a circular motion, gently apply the vaginal secretion over the surface of the pH test paper. 2. Immediately observe the color reaction on the paper and compare the color to a color comparison chart to determine the pH of the sample. 3. Record the results. Figure 15–1 Squamous epithelial cells identifiable under low power (×100). 3920_Ch15_269-282 23/01/14 10:40 AM Page 273 Chapter 15 | Vaginal Secretions 273 secretions of a healthy female. Clumps of epithelial cells as observed in Figure 15–2 are an indication of the presence of increased numbers of yeast.3 Clue Cells Clue cells are an abnormal variation of the squamous epithelial cell and are distinguished by coccobacillus bacteria attached in clusters on the cell surface, spreading past the edges of the cell, making the border indistinct or stippled. This gives the cell a granular, irregular appearance sometimes described as “shaggy.” Clue cells are diagnostic of bacterial vaginosis caused by G. vaginalis (Fig. 15–3). The presence of clue cells also can be found in urine sediment, and should be confirmed by the procedures already described. White Blood Cells White blood cells (WBCs) measure 14 to 16 µm in diameter and exhibit a granular cytoplasm. They are often described as polymorphonuclear white blood cells (PMNs) because of their characteristic multi-lobed nucleus (Fig. 15–4). In health, WBCs are present in rare to scanty numbers in vaginal secretions. Greater than 3+ WBCs in vaginal secretions suggest vaginal candidiasis, atrophic vaginitis, or infections with Trichomonas, Chlamydia, N. gonorrhoeae, or Herpes simplex.3 Figure 15–2 Clump of squamous epithelial cells (×400). Figure 15–3 Clue cells (×400). Figure 15–4 White blood cells. Notice the multilobed nucleoli (×400). Red Blood Cells Red blood cells (RBCs) appear as smooth, non-nucleated biconcave disks measuring approximately 7 to 8 µm in diameter (Fig. 15–5). RBCs can be somewhat distorted in vaginal specimens. RBCs are not usually seen in vaginal secretions, but they might be present during menstruation or due to a desquamative inflammatory process. 2 RBCs can be confused with yeast cells and are distinguished from yeast cells by KOH, which will lyse the RBCs but allow the yeast cells to remain intact. Parabasal Cells Parabasal cells are round to oval shaped and measure 16 to 40 µm in diameter. The nucleus to cytoplasm ratio is 1:1 to 1:2, with marked basophilic granulation or amorphic basophilic structures (“blue blobs”) in the surrounding cytoplasm. They are located in the luminal squamous epithelium of the vaginal mucosa. It is rare to find parabasal cells in vaginal secretions but less mature cells may be found if the patient is menstruating and in postmenopausal women.2 Increased numbers of parabasal cells, if present with large numbers of WBCs, can indicate desquamative inflammatory vaginitis2 (Fig. 15–6). Figure 15–5 Normal red blood cells (×400). 3920_Ch15_269-282 23/01/14 10:40 AM Page 274 274 Part Three | Other Body Fluids A Figure 15–6 Parabasal cell surrounded by epithelial cells (×400). Basal Cells Basal cells are located deep in the basal layer of the vaginal stratified epithelium. These cells are round and measure 10 to 16 µm in diameter and have a nucleus to cytoplasm ratio of 1:2. Basal cells are distinguished from WBCs that are similar in size by their round rather than lobed nucleus. They are not normally seen in vaginal fluid, and if present and accompanied by large numbers of WBCs and altered vaginal flora can suggest desquamative inflammatory vaginitis.2 Bacteria The vagina is a nonsterile environment with complex endogenous bacterial flora that varies with the age and hormonal status of the patient. Lactobacillus spp. normally comprise the largest portion of vaginal bacteria.7 They appear as large gram-positive, nonmotile rods on Gram stain and produce lactic acid, which maintains the vaginal pH at 3.8 to 4.5 (Fig. 15–7 A and B). Hydrogen peroxide, produced by lactobacilli subgroups, can also help to suppress the overgrowth of other organisms. Other bacteria commonly present include anaerobic streptococci, diphtheroids, coagulase-negative staphylococci, and ␣ -hemolytic streptococci. When conditions are present that cause an imbalance in the normal flora, vaginitis can occur. Absent or decreased numbers of lactobacilli relative to the number of squamous epithelial cells suggests an alteration in the normal flora. The lactobacilli are often replaced by increased numbers of Mobiluncus spp. (thin, curved, gram-negative, motile rods), Prevotella spp., Porphyromonas spp., Bacteroides spp. (anaerobic gram-negative rods), Gardnerella vaginalis (short, gram-variable coccobacilli), Peptostreptococcus spp. (gram-positive cocci), Enterococcus spp. (gram-negative cocci), Mycoplasma hominis, and Ureaplasma urealyticum. Trichomonas vaginalis Trichomonas vaginalis is an atrial flagellated protozoan that can cause vaginal inflammation and infection in women. The organism is oval shaped, measures 5 to 18 µm in diameter, and has four anterior flagella and an undulating membrane that extends half the length of the body.8 An axostyle bisects the B Figure 15–7 Bacteria. A, Large rods characteristic of Lactobacilli, the predominant bacteria in normal vaginal secretions (×400). B, Bacteria with white blood cells (×400). trophozoite longitudinally and protrudes from the posterior end, which enables the organism to attach to the vaginal mucosal and cause tissue damage (Fig. 15–8). The “jerky” motion of the flagella and undulating membrane characteristic of T. vaginalis can be observed in a wet mount. Care must be taken to not confuse T. vaginalis with sperm, which only have a single tail, a much smaller head (approximately one half the diameter of a RBC), and no axostyle. In addition, nonmotile trichomonads can be mistaken for WBCs (Fig. 15–9). T. vaginalis organisms quickly lose their viability after collection. Specimens must be examined as soon as possible or, if necessary, maintained at room temperature for a maximum of 2 hours before preparing the wet mount to observe the organism’s motility. Trichomonas also can be seen in a urinary microscopic sample, but cannot be reported unless motility is observed, either in movement across the slide or just in the tail. A dead trichomonad tends to appear oval and slightly larger than a WBC. Yeast Cells Candida albicans and non–Candida spp. cause most fungal infections but an occasional yeast in vaginal secretions is considered part of the normal flora. Yeast cells appear on 3920_Ch15_269-282 23/01/14 10:41 AM Page 275 Chapter 15 | Vaginal Secretions 275 Figure 15–8 Trichomonas vaginalis. (From Leventhal and Cheadle, Medical Parasitology, 6th edition, 2012, F.A. Davis Company, Philadelphia, with permission.) Figure 15–9 Trichomonas vaginalis in wet mount. (From Leventhal and Cheadle, Medical Parasitology, 6th edition, 2012, F.A. Davis Company, Philadelphia, with permission.) a wet mount as both budding yeast cells (blastophores) (Fig. 15–10) or as hyphae, which are long filaments that grow and form a mycelium (Fig. 15–11). Pseudohyphae, multiple buds that do not detach and form chains, can also be seen. It can be difficult to distinguish yeast cells from RBCs on a wet mount because both measure about 7 to 8 µm in diameter; however, differentiation can be made using the KOH test. Yeast cells stain gram positive. KOH Preparation and Amine Test The KOH slide is prepared and the amine (Whiff) test is performed by placing a drop of the saline specimen prepared Figure 15–10 Budding yeast cells (×400). from the collection swab onto a properly labeled clean slide and adding one drop of 10% KOH solution. The slide is immediately checked for a “fishy” amine odor. The result is reported as positive (presence of fishy odor) or negative (absence of fishy odor). Increased numbers of anaerobic bacteria in the vagina produce polyamines that are released into the vaginal fluid. The odor results from the volatilization of amines when the KOH is added. Because volatile amines are not present in normal vaginal secretion, a positive test result 3920_Ch15_269-282 23/01/14 10:41 AM Page 276 276 Part Three | Other Body Fluids PROCEDURE 15-3 KOH Preparation2 Figure 15–11 Yeast cells showing mycelial forms (×400). suggests bacterial vaginosis caused by increased numbers of G. vaginalis in conjunction with Mobiluncus spp. and with T. vaginalis. After the amine test has been performed, place a cover slip over the specimen, taking care to exclude air bubbles. Allow the KOH preparation to rest for 5 minutes to dissolve epithelial and blood cells. Heat may be applied to speed the dissolving process. One drop of 10% glycerin may be added after the KOH to prevent specimen deterioration.2 The slide is examined under low power (100× magnification) for the presence of yeast pseudohyphae and under high dry power (400× magnification) to identify smaller blastospores (yeast cells).2 Other Diagnostic Tests Although the wet mount and KOH slide examinations and the amine test are commonly used to diagnose bacterial vaginosis, other tests may be required for a confirmatory diagnosis. These include specimen culture, DNA probe testing, Gram stain, proline aminopeptidase assay, and point of care test kits. Gram Stain The Gram stain is considered the gold standard in identifying the causative organisms for bacterial vaginosis. It also provides PROCEDURE 15-2 Saline Wet Mount2 1. Prepare a clean glass slide labeled with the patient’s name and unique identifier. 2. Place one drop of vaginal specimen on the slide. 3. Cover the slide with a cover slip, removing any air bubbles. 4. Examine the slide with the 10× objective for epithelial cells and any budding yeast cells or pseudohyphae. 5. Examine the slide with the 40× objective and quantify organisms and cells per hpf. 6. Record the results. 1. Prepare a clean glass slide labeled with the patient’s name and unique identifier. 2. Place one drop of specimen on the slide. 3. Add one drop of 10% KOH to the slide. 4. Allow the KOH slide preparation to rest for up to 5 minutes to allow cellular tissue and other debris to dissolve. Gentle heating may speed the dissolving process. 5. Cover the specimen with a cover slip, removing any air bubbles. 6. Examine the slide under the 10× objective for overall assessment and for yeast pseudohyphae. 7. Switch to the 40× objective to examine for budding yeast cells (smaller blastospores). 8. Record the results. PROCEDURE 15-4 Amine (Whiff) Test 1. Apply one drop of the saline vaginal fluid suspension to the surface of a clean glass slide. 2. Add one drop of 10% KOH directly to the vaginal sample. 3. Holding the slide in one hand, gently fan above the surface of the slide with the other hand and assess for the presence of a fishy amine odor. 4. Report as positive or negative. Positive: The presence of a fishy odor after adding KOH. Negative: The absence of a fishy odor after adding KOH. a permanent record of the patient specimen. A scored Gram stain system is a weighted combination of the following morphotypes: Lactobacillus acidophilus (large gram-positive rods), G. vaginalis and Bacteroides spp. (small gram-variable or gramnegative rods), and Mobiluncus spp. (curved gram-variable rods). The types of bacterial morphophytes are evaluated and scored. For example, lactobacillus morphophytes are the predominant bacteria in normal vaginal flora; therefore, if 4+ lactobacillus morphophytes are present on Gram stain, and Gardnerella and Bacteroides spp. morphophytes, and curved gram-variable rods are absent, the score is 0. As indicated in Table 15–4, a Nugent score of 0 to 3 is considered normal vaginal flora, whereas a score of 4 to 6 is reported as intermediate, and a score of 7 or more is diagnostic of bacterial vaginosis. 3920_Ch15_269-282 23/01/14 10:41 AM Page 277 Chapter 15 | Vaginal Secretions Table 15–4 Lactobacillus 4+ 3+ 2+ 1+ 0 277 Nugent’s Gram Stain Criteria to Diagnose Bacterial Vaginosis Gardnerella and Bacteroides spp. Morphophytes Curved Gram-Variable Rods 0 1+ 2+ 3+ 4+ 0 1+ or 2+ 3+ or 4+ Points 0 1 2 3 4 Note: Points are added according to the morphotypes seen. Add the points for all three columns for a final sum. A score of 7 or higher indicates bacterial vaginosis.6 Culture Culture, using various types of media, is the gold standard test for detecting yeast and Trichomonas; however, it is more time consuming and requires up to 2 days for a result. Culture for G. vaginalis is not diagnostic for bacterial vaginosis because it is part of the normal flora in 50% of healthy women. Special media called Diamond’s medium is required for T. vaginalis. A commercial transport and culture pouch system for the detection of Trichomonas is now available (InPouch TV, Biomed Diagnostics, White City, OR). The specimen must be inoculated into the pouch within 30 minutes of collection and then is incubated for 5 days at 37°C in a CO2 atmosphere. The pouch is examined microscopically daily for motile trichomonads. DNA Testing DNA hybridization probe methods have been developed to specifically identify the causative pathogen for vaginitis. A DNA probe testing system, Affirm VPIII (Becton, Dickinson, Franklin Lakes, NJ), is available for differential diagnosis of G. vaginalis, Candida spp., and T. vaginalis. It is easy to perform and results are available in 1 hour with a sensitivity of 95%. This test is significantly more sensitive than wet mount microscopy and is less subjective to personnel bias compared with traditional microscopic tests. Trichomonas can also be detected by DNA probes amplified by polymerase chain reaction (PCR). Enzymes are added to the specimen that amplifies specific regions of T. vaginalis’ DNA by PCR. The number of DNA fragments is then calculated. This is the most accurate diagnostic method and it has the advantage of detecting nonviable organisms.9 Point of Care Tests Various rapid diagnostic tests are available to quickly screen for the causative agents of vaginitis and they provide a higher sensitivity and specificity for the organism sought. For example, proline aminopeptidase activity in vaginal secretions can be detected by rapid antigen tests to identify G. vaginalis.6 The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA) is an immunochromatographic strip test that detects T. vaginalis antigen from vaginal swabs in 10 minutes. The test is performed by placing the vaginal swab in the kit’s sample buffer. The trichomonas proteins are solubilized into the buffer. The test stick coated with anti-trichomonas antibodies is placed into the sample mixture. The solution migrates up the stick and if trichomonas antigens are present, they will react with the antibodies on the stick. A visible blue line and a red internal control line indicate a positive result. The OSOM BVBLUE test (Genzyme Diagnostics, Cambridge, MA) detects vaginal fluid sialidase, an enzyme produced by the bacterial pathogens associated with bacterial vaginosis, such as Gardnerella, Bacteroides, Prevotella, and Mobiluncus. The test takes 1 minute to perform and is read by examining the change in the color of the solution: blue or green is positive, yellow is negative. Commercial tests to measure an elevated vaginal pH (VS-Sense Pro Swab) and the presence of amines (FemExam pH and Amines TestCard, Litmus Concepts, Inc., Santa Clara, CA) use pH indicators and an amine test system that is read visually to identify bacterial vaginosis and trichomonas. Vaginal Disorders Bacterial Vaginosis Bacterial vaginosis (BV) is the most common cause of vaginitis, affecting 40% to 50% of women of childbearing age.1 BV occurs when there is an imbalance in the ratio of normal vaginal bacterial flora. The predominant organism in the vaginal flora is lactobacilli, which produce lactic acid that maintains the vaginal pH between 3.8 and 4.5. Additionally, certain subsets of lactobacilli produce hydrogen peroxide, which prevents the overgrowth of normal vaginal flora. As the vaginal pH becomes alkaline, lactobacilli are replaced by an overgrowth of G. vaginalis, Mobiluncus spp., Prevotella spp., Porphyromonas, Peptostreptococcus, Mycoplasma hominis, and Ureaplasma spp. The malodor and increased abnormal vaginal discharge result from this mix of organisms and is more apparent after intercourse.10 BV is associated with new or multiple sex partners, frequent douching, use of intrauterine devices, pregnancy, and a lack of the protective lactobacilli.1 There is evidence that BV is a risk factor for the premature rupture of membranes and preterm labor for pregnant women. Additional complications include pelvic inflammatory disease and endometritis as well 3920_Ch15_269-282 23/01/14 10:41 AM Page 278 278 Part Three | Other Body Fluids as an increased risk for acquiring some sexually transmitted infections (STIs) such as HIV, N. gonorrhoeae, C. trachomatis, and HSV-2.1,5 BV is diagnosed by examining the vaginal secretions for abnormal appearance or quantity, performing the pH and amine tests, and microscopically observing the wet mount for the presence of clue cells and the absence of WBCs and lactobacilli morphotypes. According to Amsel’s Diagnostic Criteria, three of the following four features must be present for the diagnosis of BV: (1) thin, white, homogeneous discharge, (2) vaginal fluid pH greater than 4.5, (3) a positive amine (Whiff) test, and (4) presence of clue cells on microscopic examination.5 The Gram stain is the gold standard for determining the ratio of each bacterial morphotype and offers a definitive diagnosis. Other tests used to diagnose BV include the DNA hybridization probe test, Affirm VPIII (Becton, Dickinson), which detects G. vaginalis, a proline aminopeptidase test (Pip Activity TestCard, Quidel, San Diego, CA), and the OSOM BVBLUE test (Genzyme Diagnostics, Cambridge, MA). Treatment is recommended for women to relieve vaginal symptoms and reduce the risk of infection. In addition, benefits of treatment include reducing the risk of acquiring an STI and reducing the risks associated with pregnancy. The recommended treatments are metronidazole (Flagyl), metronidazole gel, or clindamycin cream.5 Trichomoniasis Trichomoniasis is caused by the parasitic protozoon T. vaginalis. The infection is transmitted by sexual intercourse and causes vaginitis in women and sometimes urethritis in men. Most men are asymptomatic carriers. The infection is classified as an STI, frequently occurs with gonorrhea and Chlamydia infections, and has been associated with enhanced transmission rates of HIV.5,7,10 In pregnant women, a correlation has been found between a T. vaginalis infection and low birth weight, premature rupture of membranes, and preterm delivery.10 Trichomoniasis is characterized by a green-to-yellow frothy vaginal discharge, malodor, pruritus, irritation, dysuria, dyspareunia, and vaginal mucosa erythema, although some patients are asymptomatic. Patients may present with a “strawberry cervix” because of punctuate hemorrhages.10 Males are usually asymptomatic or may present with urethritis. Trichomoniasis can usually be diagnosed with the wet mount examination and microscopically, visualizing the motile trichomonads in a fresh specimen; however, this method has a sensitivity of only 60% to 70%. 5 The test must be performed within 2 hours of specimen collection to preserve the viability of the organism. WBCs and lactobacilli bacteria also are present with a T. vaginalis infection. The vaginal pH is greater than 4.5 and the amine test from the KOH preparation will be positive. If the wet mount is negative for motile trichomonads, a culture using Diamond’s medium or the commercially available pouch system (InPouch TV, Biomed Diagnostics, White City, OR) is recommended for detection of T. vaginalis. The DNA probe test system, Affirm VPIII (Becton, Dickinson, Franklin Lakes, NJ), and the point of care rapid antigen detection test, OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA), are available with increased sensitivity and specificity for T. vaginalis. The recommended treatment for trichomoniasis is metronidazole. For patients who develop an allergy to metronidazole or for whom treatment is not effective, a newer drug, tinidazole, is available. All sexual partners of patients, even if asymptomatic, should be treated to avoid reinfection. Candidiasis Vulvovaginal candidiasis is caused by an infection with the yeast Candida. It is a common cause of vaginitis, and nearly 75% of adult women have at least one yeast infection in their lifetime.10 Most yeast infections are caused by C. albicans, but other nonalbican species such as C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei have been isolated as the cause. Candida is part of the normal vaginal flora and an infection occurs when there is a change in the vaginal environment that permits the overgrowth of Candida and symptoms of the infection to occur. Conditions that can cause a change in the vaginal environment include the use of broad-spectrum antibiotics, oral contraceptives, or estrogen replacement therapy; hormonal changes that occur with pregnancy; ovulation; and menopause. Increased infection rates occur in immunocompromised patients and with conditions such as diabetes mellitus, iron deficiency, and HIV infection. The infection is predominantly found in women of childbearing age, who are producing large amounts of estrogen. Estrogen causes the vagina to mature and produce glycogen, which facilitates the growth and adherence of C. albicans.10 Typical clinical symptoms of vulvovaginal candidiasis include genital itching or burning, dyspareunia, dysuria, and the presence of an abnormal thick, white, curd-like vaginal discharge. The pH of the vaginal fluid remains normal (3.8 to 4.5) and the amine test is negative. In vulvovaginal candidiasis, the microscopic examination of the saline and KOH wet prep and Gram stain will reveal budding yeast and pseudohyphae forms, large numbers of WBCs, lactobacilli, and large clumps of epithelial cells. A culture and DNA hybridization probe (Affirm VPIII Microbial Identification System; Becton, Dickinson) analysis can be performed to confirm the clinical diagnosis and to identify the species of Candida. Yeast infections are treated with over-the-counter or prescription azole antifungal agents. They may be intravaginal, suppository, or oral agents, and the regimen depends on the medication. Over-the-counter intravaginal medications include butoconazole, clotrimazole, tioconazole, and miconazole. For patients with recurrent infections (four or more episodes per year), a prescription medication using a longer treatment regimen with either oral fluconazole or intravaginal butoconazole, nystatin, and terconazole may be more effective.5 Vulvovaginal candidiasis is not acquired through sexual intercourse, so treatment of sexual partners is not indicated. 3920_Ch15_269-282 23/01/14 10:41 AM Page 279 Chapter 15 | Vaginal Secretions Desquamative Inflammatory Vaginitis Desquamative inflammatory vaginitis (DIV) is a syndrome characterized by profuse purulent vaginal discharge, vaginal erythema, and dyspareunia.3 There is a heterogeneous group of causes of DIV; however, β -hemolytic gram-positive streptococci can be cultured from most patients.3 The syndrome also can occur secondary to atrophic vaginitis in postmenopausal women as a result of decreased estrogen. The vaginal secretion pH is greater than 4.5 and the amine test is negative. Wet mount and Gram stain microscopic examination of the vaginal secretions reveal large numbers of WBCs, RBCs, occasional parabasal and basal cells, squamous epithelial cells, and reduced or absent lactobacilli that have been replaced by gram-positive cocci (see Table 15–1). DIV is treated with 2% clindamycin.3 Hormone replacement therapy is effective for patients with DIV secondary to atrophic vaginitis. Atrophic Vaginitis Atrophic vaginitis is a syndrome found in postmenopausal women. This syndrome is caused by thinning of the vaginal mucosa because of reduced estrogen production and decreased glycogen production. As a result, the vaginal environment changes and the balance of normal flora is altered. Clinical symptoms include vaginal dryness and soreness, dyspareunia, inflamed vaginal mucosa, and purulent discharge. The vaginal secretion pH is greater than 4.5 and the amine test is negative. Microscopic evaluation is similar to that for DIV and includes large numbers of WBCs and the presence of RBCs, occasional parabasal and basal cells, squamous epithelial cells, and decreased numbers of lactobacilli that have been replaced by gram-positive cocci and gram-negative rods.3 Treatment of atrophic vaginitis is estrogen replacement. Topical vaginal ointments are initially used; however, for frequent recurrent episodes of atrophic vaginitis, oral or transcutaneous (patch) modes are more effective. Additional Vaginal Secretion Procedures As noted previously in this chapter, complications from vaginitis syndromes can include premature rupture of fetal membranes and a high risk of preterm labor. The fetal fibronectin enzyme test and the AmniSure (AmniSure International, LLC) test to detect the amniotic fluid protein PAMG-1 are tests to evaluate these conditions. The fern test described in Chapter 13 is another test that determines the presence of amniotic fluid in vaginal secretions. Patient history, a vaginal pH greater than 7.0, and a positive fern test are strong indicators of amniotic sac rupture. Fetal Fibronectin Test Preterm delivery, defined as delivery before the completion of 37 weeks’ gestation, is the leading cause of neonatal mortality 279 and morbidity in the United States.11 Fetal fibronectin (fFN) is an adhesive glycoprotein in the extracellular matrix at the maternal and fetal interface within the uterus. It is elevated during the first 24 weeks of pregnancy but then diminishes. The presence of fetal fibronectin in vaginal secretions between 24 and 34 weeks’ gestation is associated with preterm delivery. The test can be used by health-care providers as a means to better manage patient care and can be performed routinely as part of a prenatal visit for asymptomatic women between 22 and 30 weeks’ gestation or in symptomatic pregnant women between 24 and 34 weeks. Symptoms of preterm delivery include a change in vaginal secretions, vaginal bleeding, uterine contractions, abdominal or back discomfort, pelvic pressure, and cramping. The specimen is obtained by rotating the swab provided in the specimen collection kit across the posterior fornix of the vagina for 10 seconds to absorb the vaginal secretions. The swab must not be contaminated with lubricants, creams, soaps, or disinfectants that may interfere with the antibody-antigen reaction in the test system. The methods for detection of the fetal fibronectin enzyme immunoassay are solid-phase enzyme-linked immunosorbent assay (ELISA) or lateral flow, solid-phase immunochromatographic assay using the Rapid fFN cassette. In the fFN enzyme immunoassay, the vaginal sample is incubated with FDC-6, a monoclonal antibody specific for fFN, and the presence or absence of the fFN is determined spectrophotometrically at a wavelength of 550 nm. The rapid fFN assay is a qualitative test for the detection of fFN that uses a Rapid fFN cassette kit and a TLiIQ analyzer. The specimen swab is placed into an extraction buffer and filtered with a plunger filter. The filtered sample is dispensed onto the sample application well of the Rapid fFN cassette. The sample flows from an absorbent pad across a nitrocellulose membrane by capillary action through a reaction zone containing murine monoclonal anti-fFN antibody conjugated to blue microspheres. The monoclonal antibody FDC-6 is specific for fFN. The conjugate, embedded in the membrane, is mobilized by the flow of the sample. The sample then flows through a zone containing goat polyclonal anti-human fibronectin antibody that captures the fibronectin-conjugate complexes. The remaining sample flows through a zone containing goat polyclonal anti-mouse IgG antibodies, which captures unbound conjugate, resulting in a control line. After 20 minutes, the intensities of the test line and control line are interpreted with the TLiIQ analyzer. The results are reported as positive or negative.12 Symptomatic pregnant women with a positive fFN test are at increased risk for delivery in less than or equal to 7 to 14 days from specimen collection and asymptomatic pregnant women are at increased risk for delivery in less than or equal to 34 weeks and 6 days of gestation.12 AmniSure Test The risk of premature delivery also may be caused by the rupture of fetal membranes. In addition, rupture of fetal membranes can cause infection, fetal distress, prolapse of the umbilical cord, 3920_Ch15_269-282 23/01/14 10:41 AM Page 280 280 Part Three | Other Body Fluids postnatal endometritis, and placental abruption. A symptom of fetal membrane rupture is leakage of amniotic fluid. PAMG-1 is present in high levels in amniotic fluid and low levels in blood; therefore, it is a reliable marker of fetal membrane rupture. A normal level of PAMG-1 in pregnant women ranges from 0.05 to 0.22 mg/mL, which might increase to 3 mg/mL when vaginitis is present. Fetal membrane rupture causes increased concentrations of amniotic fluid in the vaginal secretions and can raise the PAMG-1 levels to 2,000 to 25,000 mg/mL.13 The AmniSure ROM test (AmniSure International, LLC) is a qualitative rapid test that uses an immunochromatographic device. A sample of vaginal secretions collected with a swab is placed into a vial with solvent. The swab is rotated for 1 minute to enable the solvent to extract the sample from the swab and then discarded. The AmniSure test strip is placed into the vial. Monoclonal antibodies with colloidal gold particles attached are located on the pad region of the test strip. The antibodies attach to the PAMG-1 in the sample and transport it to the test region. The solution flows from the pad region of the strip to the test region. The test region of the test strip has antibodies immobilized on it. If PAMG-1 is present in the patient sample, it will bind with antibodies in the test region, producing a line in the test region. This line is produced by gold dye attached to conjugated antibodies and indicates a rupture of fetal membranes. The second control line is designed to indicate that the test is functioning well. The test is read immediately or within 10 minutes. A positive test result indicates a membrane rupture and is indicated by the presence of two lines. When only the control line is present, it is reported as negative for membrane rupture. The test should be performed immediately after collection, but if there is a delay in testing, the specimen can be maintained in a closed sample vial and refrigerated for 6 hours. The AmniSure test quickly identifies patients with fetal membrane rupture, and appropriate intervention can take place. Log on to www.fadavis.com/strasinger for additional content related to this chapter. References 1. Egan, MA, and Lipsky, MS: Diagnosis of vaginitis, Am Fam Physician 62(5):1095–1104, Sep 2000. Web site: http:// www.aafp.org/afp/2000/0901/p1095.html 2. Clinical and Laboratory Standards Institute. Provider-Performed Microscopy Testing: Approved Guideline, ed. 2. CLSI document POCT10-A2. CLSI, Wayne, PA, 2011. 3. Metzger, GD: Laboratory diagnosis of vaginal infections. Clin Lab Sci 11:47–52, 1998. 4. Woods, GL, and Croft, AC: Specimen collection and handling for diagnosis of infectious diseases. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods, ed 22. Elsevier Saunders, Philadelphia, 2011. 5. Centers for Disease Control and Prevention: Diseases characterized by vaginal discharge. Sexually Transmitted Diseases Treatment Guidelines, 2010. http://www.cdc.gov/std/ treatment/2010/vaginal-discharge.htm 6. French, L, Horton, J, and Matousek, M: Abnormal vaginal discharge: Using office diagnostic testing more effectively, J Fam Practice 53(10): Oct 2004. Web site: http://www. jfponline.com/Pages.asp?AID=1788 7. Fader, RC: Anaerobes of clinical importance. In Mahon, CR, Lehman, DC, and Maneselis, G: Textbook of Diagnostic Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO, 2011. 8. Smith, LA: Diagnostic Parasitology. In Mahon, CR, Lehman, DC, Maneselis, and Maneselis, G: Textbook of Diagnostic Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO, 2011. 9. Patil, MJ, Magamoti, JM, and Metgud, SC: Diagnosis of Trichomonas vaginalis from vaginal specimens by wet mount microscopy, in pouch TV culture system, and PCR. J. Global Infect Dis [serial online] [cited 2012 Jul 6] 4:22–25, 2012. Web site: http://www.jgid.org/text.asp?2012/4/1/22/ 93756 10. Keen, EF, and Aldous, WK: Genial infections and sexually transmitted diseases. In Mahon, CR, Lehman, DC, Maneselis, G: Textbook of Diagnostic Microbiology, ed 4, Saunders Elsevier, Maryland Heights, MO, 2011. 11. Lockwood, CJ, Senyei, AE, Dische, MR, Casal, DC, et al: Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery. New Engl J Med 325:669–674, 1991. 12. Fetal Fibronectin Enzyme Immunoassay and Rapid fFN for the TLiIQ System. AW-04196-002 Rev.002, Hologic, Inc. Web site: http://www.ffntest.com/;hcp/science_fetal.html 13. Cousins, LM, et al: AmniSure Placental Alpha Microglobulin-1 Rapid Immunoassay versus standard diagnostic methods for detection of rupture of membranes. Am J Perinatol 22(6): 317–320, Aug 2005. 3920_Ch15_269-282 23/01/14 10:42 AM Page 281 Chapter 15 | Vaginal Secretions 281 Study Questions 1. Which of the following would not be a reason to collect a vaginal fluid for analysis? A. Vaginitis B. Complications of pregnancy resulting in preterm delivery C. Forensic testing in a sexual assault D. Pregnancy testing 2. Which of the following organisms might not be detected if the specimen for vaginal secretion analysis had been refrigerated? A. Prevotella bivia B. Lactobacillus acidophilus C. Trichomonas vaginalis D. Candida albicans 3. The appearance of the vaginal discharge in vulvovaginal candidiasis is described as: A. Clear and colorless B. Thin, homogeneous white-to-gray discharge C. White, curd-like D. Yellow-green and frothy 4. A normal range for a vaginal pH is: A. 3.8 to 4.5 B. 5.0 to 6.0 C. 6.0 to 7.0 D. 7.0 to 7.4 5. Which of the following tests differentiates budding yeast cells from RBCs? A. pH B. Saline wet mount C. KOH prep D. Whiff test 6. Which of the following constituents is normal in healthy vaginal fluid secretions? A. Lactobacilli B. Basal cells C. Trichomonas vaginalis D. Pseudohyphae 7. Vaginal specimens collected for a saline wet prep should be: A. Refrigerated to preserve motility B. Prepared as soon as possible C. Mailed to a reference laboratory D. Preserved with potassium hydroxide 8. A positive amine (Whiff) test is observed in which of the following syndromes? A. Bacterial vaginosis B. Vulvovaginal candidiasis C. Atrophic vaginitis D. Desquamative inflammatory vaginitis 9. A squamous epithelial cell covered with coccobacilli that extends beyond the cytoplasm margin is a: A. Basal cell B. Parabasal cell C. Clue cell d. Blastospore 10. All of the following are diagnostic of bacterial vaginosis except: A. Vaginal pH of 3.8 B. Presence of clue cells C. Positive amine (Whiff) test D. Thin, homogeneous white-to-gray vaginal discharge 11. Which of the following organisms produces lactic acid and hydrogen peroxide to maintain an acid vaginal environment? A. Gardnerella vaginalis B. Mobiluncus spp. C. Lactobacilli spp. D. β -Hemolytic streptococci 12. All of the following are diagnostic of vulvovaginal candidiasis except: A. Large numbers of WBCs B. Presence of clue cells C. Positive KOH test D. Vaginal pH of 4.0 13. All of the following are diagnostic of trichomoniasis except: A. Vaginal pH of 6.0 B. Positive amine test C. Positive KOH test D. Motile trichomonads present 3920_Ch15_269-282 23/01/14 10:42 AM Page 282 282 Part Three | Other Body Fluids 14. The bacteria associated with desquamative inflammatory vaginitis is: A. β -Hemolytic streptococci B. Trichomonas vaginalis C. Gardnerella vaginalis D. Mycoplasma hominis 15. The protein present in vaginal secretions that can identify patients who are at risk for preterm delivery is: A. Human chorionic gonadotropin B. Estrogen C. PAMG-1 D. Fetal fibronectin Case Studies and Clinical Situations 1. A 30-year-old woman has symptoms of dysuria, vaginal itching, and a white, curd-like discharge. During her visit at the Women’s Clinic, the patient revealed that she had recently completed a regimen of broad-spectrum antibiotics as treatment for a urinary tract infection. Her health-care provider takes a swab of the vaginal secretions for analysis. a. What tests will be performed on the vaginal specimen? b. Based on the patient history and observation of the vaginal secretion, which test will be diagnostic for the probable diagnosis? c. What confirmatory test can be performed? d. What is the probable diagnosis? e. What is the first choice of treatment? 2. A sexually active teenager visited the Women’s Clinic complaining of vaginal itching and soreness. She indicated that she was experiencing increased vaginal secretions that were frothy and yellow to green. Upon examination, the healthcare provider noted a strawberry-like cervix and performed a pH test on the secretions. The pH was 5.5 and the wet prep demonstrated “swimming” organisms. a. What is the probable diagnosis? b. What other tests can be performed to confirm this diagnosis? c. What is the best course of treatment? d. Should her sexual partner be treated? e. List three complications that can occur with this disorder. 3. During a routine visit with the gynecologist, a 60-year-old woman complained of vaginal dryness and soreness. During the examination, the health-care provider noted erythema of the vaginal mucosa. The pH of the vaginal secretions was 6.0. The KOH and amine (Whiff) tests were negative. The microscopic examination revealed epithelial cells, basal cells, decreased lactobacilli, and increased gram-positive cocci and gram-negative rods. a. What is the name of this condition? b. Explain how this condition can occur. c. What is the treatment for this condition? 3920_App A_283-292 23/01/14 9:16 AM Page 283 APPENDIX A Urine and Body Fluid Analysis Automation Urinalysis Automation Studies have shown that the major variable in urinalysis testing is the conscientiousness of the laboratory personnel in their timing and interpretations of the color reactions. Correct color readings depend on the accuracy of the timing. The ultimate goal of urinalysis automation is to improve reproducibility and color discrimination while increasing productivity and standardization for reporting urinalysis results. Subjectivity associated with visual discrimination among colors has been alleviated by the development of automated reagent strip readers that use a spectrophotometric measurement of light reflection termed “reflectance photometry.” Reflectance photometry uses the principle that light reflection from the test pads decreases in proportion to the intensity of color produced by the concentration of the test substance. In reflectance photometry, a monochromatic light source is directed toward the reagent pads by placing a filter between the light source and the reflective surface of the pad or by using a light-emitting diode (LED) to provide the specific wavelength needed for each test pad color reaction. The light is reflected to a photodetector and an analog or digital converter. The instruments compare the amount of light reflection with that of known concentrations, then display or print concentration units or transmit data to a laboratory information system (LIS). Several automated instruments are available that standardize sample processing, analyze test strips, perform urine sediment analysis, and report results with consistent quality and reduced hands-on time. The instruments are user-friendly and include visual and audio prompts for operation. The various manufacturers’ instruments include different features and principles for testing. See Table A–1 for a breakdown of measurement technology used by the major urine analyzer manufacturers. Table A–1 Additional advantages to automation include online computer capability with LIS interface; bar coding; manual entry of color, clarity, and microscopic results to be included on the printed report; flagging abnormal results; storing patient and control results; and minimal calibration, cleaning, and maintenance. Automated instruments in urinalysis include semiautomated and fully automated chemistry analyzers, automated urine cell analyzers, and completely automated systems. Semi-automated instruments still depend on an operator for specimen mixing, test strip dipping, and microscopic results input. In a fully automated chemistry analyzer, the tubes of urine are placed on a rack or a carousel and moved automatically through the instrument. Automated urine cell analyzers mix, aspirate, dilute, and stain urine to classify urine sediment particles. Automated urine systems perform a complete urinalysis that includes the physical, chemical, and microscopic parts of a routine urinalysis. The automated urinalysis instruments currently available are listed in Table A–2; however, new instruments are continually being developed. Semi-Automated Urine Chemistry Analyzers Semi-automated urine analyzers test for the chemical components of urine. The instruments read and interpret the reagent strip results consistently, thereby standardizing the interpretation of reagent strip results and eliminating personnel color bias and timing discrepancies. Depending on the instrument and the reagent strip used, the following tests can be performed: leukocyte, nitrite, protein, blood, glucose, ketone, bilirubin, urobilinogen, pH, specific gravity, color, creatinine, and protein-to-creatinine ratio. Semi-automated analyzers are well suited for small- and medium-volume laboratories and Measurement Technology Methods in Automated Urinalysis Urine Measurement Technology Manufacturer Color Clarity Specific Gravity ARKRAY, Inc Iris Diagnostics Roche Diagnostics Siemens Healthcare Diagnostics, Inc. Photometry Light transmission/light scatter Reflectance photometry Reflectance photometry Light scatter Light transmission/light scatter Turbidity Light transmission/light scatter Refractive index Refractive index Refractometry Refractive index 283 3920_App A_283-292 23/01/14 9:16 AM Page 284 284 Appendix A | Urine and Body Fluid Analysis Automation Table A–2 Urinalysis Automation Equipment Manufacturer Semiautomated Chemistry Instruments Clinitek Advantus Siemens Healthcare Diagnostic, Inc. Clinitek Status Siemens Healthcare Diagnostics, Inc. Urisys 1800 system Roche Diagnostics COBAS u411 Roche Diagnostics DiaScreen 50 U. S. ARKRAY iChem 100 Iris Diagnostics Fully Automated Chemistry Instruments Clinitek Atlas Siemens Healthcare Diagnostic, Inc. Urisys 2400 system Roche Diagnostics Aution Max AX-4030 U. S. ARKRAY IChem Velocity Iris Diagnostics Automated Microscopy UF-1000i Urine Sysmex Corporation Cell Analyzer iQ 200 Automated Iris Diagnostics Urine Microscopy Urine Analyzer (iQ Iris Diagnostics 200 Sprint) Automated Urinalysis Systems iRICELL Urinalysis Iris Diagnostics Systems (iRICELL 3000plus, iRICELL 2000plus, iRICELL 3000pro, iRICELL 2000pro, iRICELL 1500) CLINITEK AUWi Siemens Healthcare Diagnostics, Inc System and specimen color and clarity may be entered manually or a barcode reader can be used to identify samples. Positive results are flagged to indicate a patient sample that requires additional confirmation testing or microscopic evaluation. The semi-automated instrument requires the operator to: 1. Dip the reagent strip into a well-mixed urine sample. 2. Blot the strip to remove excess urine. 3. Place the strip onto the reagent strip platform. 4. Press the analyze/enter button. Results are printed out, and abnormal results are flagged automatically. As the strip moves through the instrument, reflectance readings are taken at the correct time intervals. Some manufacturers’ test strips have a color compensation pad that adjusts results for urine color. This feature allows the instrument to subtract the urine color from the color developed on the reaction pad, providing an accurate result for each pad despite the interference. The strips are then moved to the waste container. Results are stored in the analyzer, printed, or sent to the LIS. Examples of semi-automated instruments are shown in Figures A–1 through A–6. Daily maintenance is minimal and includes cleaning the reagent strip platform and emptying the reagent strip waste container. Fully Automated Urine Chemistry Analyzers Fully automated instruments are designed for a high-volume urinalysis laboratory with user walk-away capability. The various instruments have the ability to load many samples on a carousel or rack at one time with the capability to insert a STAT specimen during the run. The start or analyze button is pressed to begin testing and the specimen automatically moves through the instrument. The specimen is identified. A sample probe aspirates Body Fluid Analyzers ADVIA2120i with Body Fluids Software Sysmex XE-5000 using Body Fluids mode iQ 200 using Body Fluids Software Siemens Healthcare Diagnostics, Inc. Sysmex Corporation Iris Diagnostics physicians’ offices, and meet the Clinical Laboratory Improvement Amendments (CLIA)-waived standards. Semi-automated analyzers are self-calibrating and some instruments perform automatic checks (Auto-Checks) to identify strip type and humidity exposure. For a semi-automated instrument, the reagent strips are manually dipped and placed on the strip reader, the reaction pads are read at the correct time, and the strip is moved to the waste container. The results are displayed, printed, or transmitted to an LIS. Patient identification Figure A–1 DiaScreen50 semi-automated urine chemistry analyzer. (Image courtesy of U. S. ARKRAY.) 3920_App A_283-292 23/01/14 9:16 AM Page 285 Appendix A | Urine and Body Fluid Analysis Automation 285 A Figure A–2 Cobas u 411 urine chemistry analyzer. (Image courtesy of Roche Diagnostics.) B Figure A–4 Clintek Status + Analyzer. A, Clinitek Status Connect with Barcode Stand. B, Clinitek Status with test strip. (Images courtesy of Siemens Healthcare Diagnostics Inc.) Figure A–3 Urisys 1100 semi-automated urine chemistry analyzer. (Image courtesy of Roche Diagnostics.) Figure A–5 Clinitek Advantus semi-automated urine chemistry analyzer. (Image courtesy of Siemens Healthcare Diagnostics Inc.) 3920_App A_283-292 23/01/14 9:17 AM Page 286 286 Appendix A | Urine and Body Fluid Analysis Automation Figure A–6 iChem 100 semi-automated urine chemistry analyzer. (Image courtesy of Iris Diagnostics.) an exact amount of urine sample and dispenses it directly onto the reagent strip. The reagent strip advances automatically to the reflectance photometer to measure the color change of each reagent pad. The strip then advances automatically to the disposal area. Tests are measured by the dry pad test using reflectance photometry to detect color change, and taking readings at the appropriate time and wavelength for each specific test. Analytes measured vary by the instrument and may include leukocytes, ketones, protein, glucose, nitrite, blood, urobilinogen, pH, bilirubin, color, clarity, creatinine, and protein-low. Color is measured by either reflectance photometry or spectrophotometry at multiple wavelengths. Specific gravity is measured by refractive index methodology, and clarity is a measurement of transmitted or scattered light. The instruments use integrated bar-coded sample identification and allow abnormal ranges to be selected so that samples that require microscopic examination or confirmatory testing can be identified and flagged. Patient results and quality control results and calibrations are stored for visual display, printout, or transmission to a laboratory computer system. Standardized controls are run as set by laboratory protocol. Examples of fully automated urine chemistry analyzers are shown in Figures A–7 through A–10. Figure A–7 Urisys 2400 automated urine chemistry analyzer. (Image courtesy of Roche Diagnostics.) Automated Microscopy In a routine urinalysis, a test strip determines the chemical analytes, and the formed elements are determined by microscopy. Manual microscopy is not easily standardized because of the high variation among operators even in the same institution. Routine specimen processing such as centrifugation can affect accuracy because rare elements such as casts or cells may be lost during handling. It has been demonstrated that recovery of formed elements in the sediment after centrifugation is highly variable. Results are not quantitative because they must be reported in ranges or averages. Overall, manual microscopy is not cost effective because of the labor and time required to process and analyze the specimen, which ultimately reduces turnaround times for results. Automated urine cell analyzers provide efficient standardized results in less than 1 minute as compared with Figure A–8 Clinitek Atlas automated urine chemistry analyzer. (Image courtesy of Siemens Healthcare Diagnostics Inc.) approximately 6 minutes using the manual method, markedly improving turnaround times. Two automated urine cell analyzers are currently available in the United States: the Sysmex UF-1000i and the iQ 200 from Iris Diagnostics. Sysmex UF-1000i The Sysmex UF-1000i (Sysmex Corporation, Kobe, Japan) uses laser-based flow cytometry that measures forward light scatter, side scatter, fluorescence staining characteristics, and adaptive 3920_App A_283-292 23/01/14 9:17 AM Page 287 Appendix A | Urine and Body Fluid Analysis Automation 287 cells (WBCs), epithelial cells, hyaline casts, and bacteria. The results are displayed as scattergrams (Fig. A–15). These parameters are directly reportable without technologist intervention and may be autovalidated. Flagged particles include pathologic casts, crystals, small round cells (renal tubular epithelial cells or transitional epithelial cells), sperm, mucus, and yeast-like cells, and must be confirmed by manual microscopy. A latex particle quality control system monitors performance, and quality control records can be viewed on the instrument screen in a Levy-Jennings graph. The analyzers can store up to 10,000 patient results, including scattergrams, histograms, and specimen characteristics, which can be saved and viewed by the user. A bidirectional interface is provided to download and report results. The UF-1000i can be used independently or integrated with an automated urine chemistry strip reader to create a complete urinalysis system. Figure A–9 Aution Max AX-4030 fully automated urine chemistry analyzer. (Image courtesy of U. S. ARKRAY.) Figure A–10 iChem Velocity automated urine chemistry analyzer. (Image courtesy of Iris Diagnostics.) cluster analysis to identify stained urine sediment particles (Fig. A–11). To perform an automated microscopy, 4 mL of uncentrifuged urine is aspirated into the instrument and divided into two channels: one for urine particle analysis and one for bacteria staining and detection (Fig. A–12). Each channel has a specific stain that targets the internal components of the cells. The stained urine sample passes through the flow cell, where it is hydrodynamically focused and presented to a red semiconductor laser (635 nm) (Fig. A–13). Particles are identified by measuring the height and width of the fluorescent and light scatter signals, which are presented in scattergrams and histograms. In the bacterial channel, the diluent stabilizes the pH and lyses the nonbacterial particles, eliminating amorphous crystals. The stain is specific to the RNA in a bacterial cell, eliminating any nonspecific staining of debris. The width of the fluorescent signal measures cellular inclusions and the width of forward light scatter measures the length of cells (Fig. A–14). Resulting values are presented in quantitative cells per microliter and cells per high- or low-powered field. Thresholds to be flagged for primary elements can be established and abnormal results are flagged for confirmatory review. The main particles enumerated are red blood cells (RBCs), white blood Figure A–11 Sysmex UF 1000i urine chemistry analyzer. (Image courtesy of Sysmex Corporation, Mundelein, IL.) Forward scatter signal amplifier Fluorescence signal amplifier Red semiconductor laser Sheath reagent Side scatter signal amplifier Conductivity sensor Dilution and staining for bacteria analysis Dilution and staining for sediment analysis Urine sample Flow cytometry Figure A–12 Diagram of urine particle analysis in the Sysmex UF1000i. 3920_App A_283-292 23/01/14 9:17 AM Page 289 Appendix A | Urine and Body Fluid Analysis Automation 289 Figure A–18 Auto-Particle Recognition (APR) process. (Image courtesy of Iris Diagnostics.) Figure A–15 Scattergram showing Sysmex UF1000i microscopy results. Figure A–19 iQ 200 urinalysis results display, showing particle categories available for analysis or counting. (Image courtesy of Iris Diagnostics.) Figure A–16 iQ 200 microscopy analyzer. (Image courtesy of Iris Diagnostics.) The sedMAX (77Elektronika, distributed in Europe and Asia by A. Menarini Diagnostics) performs automated microscopy with digital imaging. The instrument uses newer technology, and as of this writing is not yet FDA approved for use in the United States. The sedMAX requires a minimum of 2 mL of urine that is centrifuged in a special cuvette to produce a monolayer of urine sediment. The sediment is analyzed by a bright-field microscope and digital camera to capture and categorize 15 particle images based upon size and shape using image-processing software.2 An advantage to this instrument is the zoom capability to view images and interpretation of the images is similar to manual microscopic smears. Automated Urinalysis Systems Figure A–17 Diagram of the iQ 200 digital flow capture process. (Image courtesy of Iris Diagnostics.) reviewed easily and reclassified by the operator without the need for manual microscopy. The software allows the user to subclassify 27 additional categories to indicate the specific types of crystals, casts, epithelial cells, yeast with pseudohyphae, trichomonads, and oval fat bodies. Free text comments can be added to the report as needed. Combining automated urine chemistry analyzers and automated urine cell analyzers to create completely automated urinalysis systems has significantly improved turnaround times for urinalysis. Technologists’ hands-on time has been significantly reduced as well. Using similar sample racks and moving on a conveyor system, samples are easily transferred from one instrument to the next, providing a complete walkaway capability with minimal specimen handling from sampling through results. By interfacing with the LIS, bar-coded samples are automatically identified and processed according to the requested tests. The systems can independently perform both physical and chemical testing, microscopy analysis, and a combination of both. A complete urinalysis report can be sent directly to the LIS or printed out, thereby reducing clerical error. The Clinitek Atlas (Siemens Healthcare Diagnostics, Deerfield, IL), an automated urine chemistry analyzer, and the 3920_App A_283-292 23/01/14 9:17 AM Page 290 290 Appendix A | Urine and Body Fluid Analysis Automation Sysmex UF-1000i (Sysmex Corporation, Mundelein, IL), an automated urine cell analyzer, have been integrated to develop the CLINITEK AUWi System (Siemens), which performs a completely automated urinalysis (Fig. A–20). A minimum of 5 mL of urine is required in the automated mode. The bar-coded tubes are racked and placed onto the system. The rack advances to the ATLAS analyzer, where the specimen is identified, mixed, aspirated, and tested for physical and chemical components. The sample then travels across a connecting bridge to the UF-1000i for microscopic analysis. The instrument automatically reflexes samples requiring sediment analysis, reducing the time associated with manual microscopic analysis. Results are automatically verified and integrated into a complete urinalysis report to be sent to the LIS or printed. The iRICELL Automated Urinalysis Systems (Iris Diagnostics) consist of the iChem VELOCITY urine chemistry analyzer and the iQ 200 (Fig. A–21). A minimum of 4 mL of urine is required. The bar-coded tubes are placed into the 10-position rack and are moved to the iChem VELOCITY, where the sample is mixed and the urine is aspirated. Upon completion of the physical and chemical analysis, the rack moves across the connecting bridge to the iQ 200 for microscopy testing. Samples can be reflexed to urine microscopy based upon urine chemistry results. One hundred percent microscopy is not required with iRICELL. Results for a complete urinalysis are transmitted to the LIS or printed. Figure A–20 AUWi, a fully automated urinalysis system that combines the Siemens Clinitek Atlas Chemistry analyzer and the Sysmex UF-1000i particle analyzer. (Image courtesy of Siemens Healthcare Diagnostics Inc.) Figure A–21 iRICELL3000, a fully automated Urinalysis System that combines the iChem Velocity urine chemistry analyzer and the iQ 200 microscopy analyzer. (Image courtesy of Iris Diagnostics.) Body Fluid Analysis Automation Body fluid cell counts and differentials are traditionally performed manually using a Neubauer hemocytometer. These are labor-intensive and time-consuming procedures subject to technologist variability. Automation brings quality control and precision to a method that previously was uncontrolled. Automated instruments have not, however, completely eliminated the use of a manual hemacytometer count. Body fluids with low cell counts or malignant cells still require a manual differential using a stained cytospin smear. The laboratory must define the limits for the instrument and establish the lower limits for cell counting to determine when a manual procedure must be performed.2 For example, the iQ 200 is linear down to zero (0 to 10,000 cells/L). Linearity can be extended during method comparison. In addition, the laboratory must follow manufacturers’ recommended procedures for special treatment required for the specific body fluid analyzed.3 Hematology analyzers that are used to perform body fluids cell counts include the ADVIA2120i (Siemens) the Sysmex XE 5000 (Sysmex) and the Beckman Coulter LH780 and UniCel DxH800 (Beckman Coulter, Inc.). The ADVIA 2120i uses flow cytometry, light scatter, and absorbance to count red and white blood cells, and perform a WBC differential that includes percentages and absolute numbers of mononuclear cells and polymorphonuclear cells on samples with greater than 20 WBC/µL. The WBC differential includes the numbers of neutrophils, lymphocytes, monocytes, and eosinophils. A cerebrospinal fluid (CSF) specimen is pretreated with CSF reagent to fix and spherize the cells. The prepared sample remains stable for 4 minutes to 4 hours when stored at 18°C to 30°C. The specimen is aspirated into the instrument and cells are differentiated and enumerated by three optical measurements. The signals are digitized and used to construct the CSF cytogram. With this system, more cells are counted, achieving increased accuracy and precision. The automated RBC, WBC, polymorphonuclear, mononuclear, and differential results are available within 1 minute of sample aspiration. In addition, the ADVIA 2120i can provide a rapid automated diagnostic test for fetal lung maturity by counting lamellar bodies in amniotic fluid. Lamellar bodies are counted in the platelet channel using high and low laser light scatter. The analyzer is approved for counting cells in pleural fluids, peritoneal fluids, and peritoneal dialysates. The Sysmex XE-5000 is the newest generation hematology analyzer that includes a dedicated body fluid mode. 4 The body fluid mode has extended cell counting to increase precision in samples with small numbers of cells. Body fluids can be analyzed without sample preparation or pretreatment. In the body fluid mode, a new software algorithm is used to count RBCs using sheath flow impedance and WBCs using side scatter and fluorescence intensity after dye-staining 3920_App A_283-292 23/01/14 9:17 AM Page 291 Appendix A | Urine and Body Fluid Analysis Automation 291 nuclear DNA or RNA. Polymorphonuclear cells and mononuclear cells are counted in the differential (DIFF) channel. Mesothelial cells and macrophages are enumerated but are not included in the cell count. It is recommended that only nonmalignant fluid be analyzed on the XE-5000. In addition, the differential scatterplots should be inspected visually to detect non-cellular particulate matter such as bacteria, Cryptococcus, and interference from large cells (macrophages and mesothelial cells). With WBC counts below 10×106/µL cells, differentiation between polymorphonuclear cells and mononuclear cells should not be done.4 The iQ 200 using digital flow cell imaging can be used for body fluids by adding the body fluids software module. The body fluid menu includes CSF, synovial, pleural, peritoneal, peritoneal dialysate, peritoneal lavage, pericardial, and general serous fluids. Two dilutions are made on the specific fluid based on the type of fluid and the appearance of the fluid. One tube is diluted with an Iris (iQ) diluent and the other with an Iris RBC-lysing reagent. The dilution-specific barcoded tubes are loaded onto the instrument for counting. A total cell count is derived from the Iris (iQ) diluent tube and the nucleated cell count is obtained from the Iris lysing reagent dilution. The difference between these two numbers equals the RBC count. As with urine microscopy, results and cell images are sent to an operator screen for review or autoverification and can be sent directly to the LIS. References 1. Laboratory Production Solutions, Iris Diagnostics, August 31, 2012. 2. Block, DR, and Lieske, JC: Automated Urinalysis in the Clinical Lab. Medical Laboratory Observer (MLO), 44(10), Oct 2012. 3. Clinical and Laboratory Standards Institute: Body fluid analysis for cellular composition: approved guideline, CLSI document H-56A. CLSI, Wayne, PA, 2006. 4. deJonge R, Brouwer R, de Graaf MT, et al: Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids. Clin Chem Lab Med 48:665–675, 2010. Additional Information Sources ARKRAY Inc., Kyoto, Japan: www.arkrayusa.com Iris Diagnostics, Chatsworth, CA: www.irisdiagnostics.com Roche Diagnostics, Indianapolis, IN: www.roche.com/products Siemens Healthcare Diagnostics Inc., Deerfield, IL: www.usa. siemens.com/diagnostics Sysmex America, Inc. Mundelein, IL: www.sysmex.com/usa 3920_App A_283-292 23/01/14 9:17 AM Page 292 3920_App B_293-296 23/01/14 9:17 AM Page 293 APPENDIX B Bronchoalveolar Lavage Analyzing specimens obtained by bronchoalveolar lavage (BAL) is a method for obtaining cellular, immunologic, and microbiologic information from the lower respiratory tract. BAL is particularly useful in evaluating immunocompromised patients, interstitial lung disease (infectious, non-infectious, immunologic, or malignant), airway diseases, suspected alveolar hemorrhage, pulmonary alveolar proteinosis, Langerhans cell histiocytosis, and dust exposure. It is often used in conjunction with high-resolution computerized tomography (HRCT), medical history, and physical examination to determine the need for a surgical biopsy. During bronchoscopy, a fiber-optic bronchoscope is guided into a selected bronchopulmonary segment, usually the right middle or lingular lobe; however, target areas are better defined using HRCT before the procedure. Optimal targets are areas of alveolar ground glass opacity, more prominent nodular profusion, or fine reticulation.1 The segment lavaged should be recorded on the requisition form. Aliquots of sterile normal saline are instilled into the alveolar spaces through the bronchoscope to mix with the bronchial contents and are aspirated for cellular examination and culture. The instillation volume is between 100 and 300 mL of sterile saline in 20- to 50-mL aliquots.2 The first aliquot is discarded, the remaining aliquots are either sent individually for analysis or pooled for further analysis.2 The desired fluid volume for analysis is 10 to 20 mL (minimal volume is 5 mL). Optimal sampling retrieves greater than 30% with a typical recovery range of 50% to 70%. Lowvolume recovery (less than 25%) caused by fluid retention in the lung may appear in chronic obstructive lung diseases and should be noted on the requisition form. Keep specimens at room temperature during transport to the laboratory and process them immediately. When delivery to the laboratory is delayed for longer than 30 minutes, transport the specimens on ice (4°C).1 Specimens that will not be analyzed immediately should be centrifuged, the cells resuspended in a nutrient-supplemented medium, and refrigerated at 4°C for up to 24 hours.1 Specimens are unacceptable for testing after 24 hours. Cell counts should be performed within 1 hour or are stable for up to 3 hours if the fluid is in a nutrient-supplemented medium.2 Samples can be filtered through loose gauze (50 to 70 µm nylon filter) to remove mucus, phlegm, and dust. Diagnostic tests on BAL fluid include a cell count with differential, microbiologic studies, and cytopathology. A macroscopic observation is recorded describing the color and clarity of the specimen. The appearance of the BAL fluid can provide valuable diagnostic information. BAL fluid color can be clear (colorless), milky white, light brown-beige, and red. BAL fluid clarity may be described as clear, hazy, cloudy, or turbid. A bloody BAL fluid with increasing intensities during the sequential aliquots indicates acute diffuse alveolar hemorrhage, whereas orange-red BAL fluid is the result of an older hemorrhagic syndrome and would be evaluated for intracellular iron content by cytochemistry.2 A milky or light brown-beige color BAL fluid indicates an accumulation of phospholipid-protein complexes derived from pulmonary surfactant in the lung alveoli and strongly suggests pulmonary alveolar proteinosis. The BAL fluid should be centrifuged if it looks milky.2 The presence of clots should be noted. The fluid volume is measured and cell counts and differential counts are performed. White and Red Blood Cell Counts White blood cell (WBC) and red blood cell (RBC) counts are performed on BAL and may be diluted to facilitate counting using a hemocytometer. Cell viability can be determined by adding Trypan blue. Counts also can be performed with certain automated cell counters as designated by the manufacturer. When cell concentration is less than the automated instrument’s linearity specifications, hemocytometry should be used.2 If a hemocytometer is used, WBC counts may be diluted using the BMP LeukoChek system to facilitate counting. A BMP LeukoChek system is available with a 1 to 100 dilution of ammonium oxalate to lyse the RBCs. When the RBCs have lysed and the solution is clear, plate the fluid on a hemocytometer and allow the cells to settle for 5 minutes. Count all cells in the 18 squares on both sides of the hemacytometer and calculate the average of the two sides. Using the following formula, calculate the WBC count using the following formula:3 WBC/cmm = average number of cells × dilution factor × 10 9 squares RBC counts may be diluted with isotonic saline using an MLA pipette. Plate the fluid on a hemocytometer and allow it to settle for 5 minutes. Count both sides of the hemocytometer and use the following formula to calculate the RBC/cmm:3 RBC/cmm = number of cells × dilution factor × 10 Number of squares counted Cells must be evenly distributed over the hemocytometer surface. For a leukocyte count within the reference range, there should be no more than a 15-cell difference between the highest and lowest total number of cells found among the squares counted. For a RBC count, there should be no more than a 30-cell difference between the highest and lowest total number of cells found among the squares counted. Total cells counted 293 3920_App B_293-296 23/01/14 9:17 AM Page 294 294 Appendix B | Bronchoalveolar Lavage on each side of the counting chamber should agree within 10% of each other. Counts that do not meet this standard should not be reported. Mix the specimen well and repeat the count. If clumps of cells or clots are present, note on the requisition that the “cell count may be inaccurate due to clumps of cells and/or clots.” BAL body fluids may not be counted on the Sysmex instrumentation because of the varied types of cells present.3 Leukocytes Evaluating the predominant inflammatory cellular pattern provides valuable information to the clinician in determining a differential diagnosis. The morphologic appearance of cells and particles, such as the morphology of macrophages in extrinsic allergic alveolitis and sarcoidosis, or the detection of dust particles in occupational exposure conditions, provides diagnostic information.2 Differential slides are prepared by cytocentrifugation using routine procedures with staining (Wright-Giemsa or May Grunwald-Giemsa) and at least 300 cells but often 500 to 1000 cells are counted and classified.4 Cells seen in BAL fluid include macrophages, lymphocytes, ratio of CD4+ and CD8+ lymphocytes (CD4/CD8 ratio), neutrophils, eosinophils, ciliated columnar bronchial epithelial cells, and squamous epithelial cells. Macrophages, often containing a variety of phagocytized material, are the cells most frequently seen, in numbers ranging from 56% to 80% (Fig. B–1). Phagocytized material includes hemosiderin; golden, brown, or black pigment inclusions; or foamy cells. A predominance of macrophages containing smoking-related inclusions suggests smoking-related interstitial lung disease or pulmonary Langerhans cell histiocytosis.1 Lymphocytes, normally constituting 1% to 15% of the cell population, are increased in interstitial lung disease, drug reactions, pulmonary lymphoma, and nonbacterial infections. A lymphocyte differential count equal to or greater than 25% suggests granulomatous lung disease, whereas a lymphocyte differential count greater than 50% suggests hypersensitivity pneumonitis or nonspecific interstitial pneumonia.1 The ratio Figure B–1 Bronchoalveolar lavage: Normal macrophages and lymphocytes (×1000). of CD4 to CD8 lymphocytes further defines the disease process. An elevated CD4/CD8 indicates sarcoidosis or connective tissue disorders. A normal CD4/CD8 is associated with tuberculosis or malignancies, whereas a low CD4/CD8 indicates hypersensitivity pneumonitis, silicosis, drug-induced disease, or HIV infection. Immunologic analysis is performed by flow cytometry. Neutrophils are the primary granulocyte seen, with a normal value of less than 3%. They are elevated in cigarette smokers, and in cases of bronchopneumonia, toxin exposure, and diffuse alveolar damage. A neutrophil count equal to or greater than 50% strongly suggests acute lung injury, aspiration pneumonia, or suppurative infection.1 Eosinophils, usually less than 1% to 2% of the total cells, are elevated in asthma, drug-induced lung disease, infections (parasitic, mycobacterial, or fungal), hypersensitivity, pneumonitis, and eosinophilic pneumonia. An eosinophil differential count greater than or equal to 25% diagnoses eosinophilic lung disease.1 A mast cell differential count greater than 1% combined with a lymphocyte count greater than 50% and a neutrophil count greater than 3% strongly suggests hypersensitivity pneumonitis.1 Erythrocytes The presence of erythrocytes indicates an acute alveolar hemorrhage. Phagocytosed erythrocytes suggest that an alveolar hemorrhage has occurred within the past 48 hours, whereas hemosiderin-laden macrophages indicate an alveolar hemorrhage older than 48 hours. Epithelial Cells Ciliated columnar bronchial epithelial cells are more numerous in bronchial wash specimens than in bronchial lavage specimens because of the more vigorous washing technique. In a lavage specimen, these cells normally range from 4% to 17% (Fig. B–2). Figure B–2 Bronchoalveolar lavage: Ciliated bronchial epithelial cells; notice the eosinophilic bar (×1000). 3920_App B_293-296 23/01/14 9:18 AM Page 295 Appendix B | Bronchoalveolar Lavage 295 Fungi, Viruses, and Bacteria Fungal elements and viral inclusions may also be observed in respiratory specimens. Organisms identified include Pneumocystis carinii, Toxoplasma gondii, Strongyloides stercoralis, Legionella pneumophila, Cryptococcus neoformans, Histoplasma capsulatum, Mycobacterium tuberculosis, Mycoplasma pneumoniae, influenza A and B viruses, and respiratory syncytial virus. Quantitative or semiquantitative cultures are useful for ventilator-associated pneumonia and can diagnose the infection if the organism is identified. With the increasing concern about nosocomial infections and antibiotic-resistant microorganisms, BAL is more frequently performed on ventilator-assisted patients to detect infection and monitor antibiotic therapy. Bronchoalveolar lavage is becoming an important diagnostic test for P. carinii in immunocompromised patients. With P. carinii, characteristic amorphous material is seen microscopically under low power and organisms are visible under high power (Figs. B–3 and B–4).5 C. neoformans has become a significant opportunistic pathogen in patients with AIDS. A diagnosis of pulmonary cryptococcosis can be made by demonstrating a positive cryptococcal antigen in respiratory specimens exhibiting yeast cells that morphologically resemble C. neoformans. The extent of the cryptococcal infection correlates with the antigen titer. Figure B–4 Bronchoalveolar lavage: Characteristic cup-shaped organisms indicating P. carinii (×1000). Cytology Cytologic studies include observing sulfur granules (actinomycetes), hemosiderin-laden macrophages, Langerhans cells, cytomegalic cells, fat droplets seen in fat embolism with an Oil Red O stain, and lipid-laden alveolar macrophages using a Sudan III stain. Periodic acid Schiff staining or Oil Red O staining may be useful in diagnosing pulmonary alveolar proteinosis or aspiration.1 Dust particle inclusions indicate pneumoconioses or asbestos exposure. Specimens are evaluated by a pathologist in cytology whenever malignancy is suspected. References Figure B–3 Bronchoalveolar lavage: Amorphous material associated with P. carinii when examined under low power (×100). 1. Meyer, KC, Raghu, G, Baughman, RP, et al.: An official American Thoracic Society clinical practice guideline: The clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. Am J Respir Crit Care Med 185(9):1004–1014, May 1, 2012. doi:10.1164/rccm.201202-0320ST. 2. Clinical and Laboratory Standards Institute: Body Fluid Analysis for Cellular Composition; Approved Guideline. CLSI document H56-A. Clinical and Laboratory Standards Institute. Wayne, PA 2006. 3. Bronchoalveolar Lavage, BAL Cell Count and Differential. Methodist Hospital: Clinical Laboratory Procedure Manual. Omaha, NE, January 5, 2012. 4. Jacobs, JA, DeBrauwer, EI, et al: Accuracy and precision of quantitative calibrated loops in transfer of bronchoalveolar lavage fluid. J Clin Micro 38(6):2117–2121, 2000. 5. Linder, J: Bronchoalveolar Lavage. ASCP, Chicago, 1988. 3920_App B_293-296 23/01/14 9:18 AM Page 296 3920_Ans_297-304 23/01/14 9:15 AM Page 297 Answers to Study Questions and Case Studies and Clinical Situations Chapter 1 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. C A D A B A C D D C B A 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. B D A B C C A B D B A C 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. D D D B 2, 1, 2, 3, 2, 1 D C D C D Case Studies and Clinical Situations 1. a. Review of the procedure by a designated authority has not been documented. b. Instructions and training are not being provided to personnel performing collections. c. A safety statement about the heat produced by the reaction is not in the procedure manual. d. The bottles have not been dated and initialled. 2. a. Correct; proficiency survey tests should be rotated among personnel performing the tests. b. Accept; QC on the Clinitest tablets must only be performed when they are used to perform a test. c. Correct; documentation of technical competency should be performed on all personnel working in the section and educational qualifications assessed. 3. a. The procedure was being performed incorrectly. The correct timing of the glucose reaction was not being done. b. The technologist performing the test. QC ensures that the reagents and instrument are working properly and that the technologist is performing the test correctly. c. At the beginning of each shift, when a new bottle of reagent is opened or as stated in the procedure manual. d. Refer to the procedure manual. Check expiration dates of controls and reagents. Open and test a new bottle of control or reagents. e. When the controls are within range. 4. a. Delay in testing the specimen. b. Incident report. c. Delta check. d. Treatment of the patient will be delayed because the specimen will need to be recollected and tested. Extra expense incurred. e. Errors should be corrected as soon as possible following the institution’s policy. The original result must not be erased. Chapter 2 Study Questions 1. 2. 3. 4. 5. 6. 7. C B C A D C C 8. 9. 10. 11. 12. 13. 14. D A A B B D A 15. 16. 17. 18. 19. 20. C C A D B B Case Studies and Clinical Situations 1. a. It could have increased pH, nitrite and bacteria and decreased clarity, glucose, ketones, bilirubin, urobilinogen and WBCs, RBCs and casts. b. The specimen was refrigerated and was brought immediately to the laboratory. 2. a. It would be less clear. b. Additional epithelial cells and bacteria (making it not acceptable for a culture). 3. a. The results would be falsely decreased. b. The patient needs to collect another specimen. 4. a. Yes. b. The temperature would be lower than body temperature. c. The specimen tested was not from the defendant. d. An accurately filled out chain of custody form (COC). 297 3920_Ans_297-304 23/01/14 9:15 AM Page 298 298 Answers to Study Questions and Case Studies and Clinical Situations Chapter 3 Chapter 4 Study Questions Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. B D C D A B C D B A C D D 14. 15. 16. 17. 18. 19. 20. 21. 22. B B D B A. Beta2-microglobulin; B. Creatinine; C. Cystatin C; D. 125Iiodothalamate B 69 mL/min D D 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. B D D C C B A +0.5 D 600 mL/min C B Case Studies and Clinical Situations 1. a. 160-mg/dL to 180-mg/dL. b. Renal tubular reabsorption is impaired. 2. a. Juxtaglomerular apparatus → Angiotensinogen → Renin → Angiotensin I → Angiotensin II. b. Vasoconstriction, increased sodium reabsorption, and increased aldosterone to retain sodium. c. Production of renin decreases and, therefore, the actions of the renin-angiotensin-aldosterone system. 3. a. The physician can calculate the approximate creatinine clearance using the MDRD-IDMS-traceable formula. b. The cystatin C test and the beta2-microglobulin test serum tests. c. No. The beta2-microglobulin test requires a normal immune system and malignancies can affect the immune system; therefore, the test cannot be reliable in patients with immunologic disorders and malignancies. 4. a. Yes. Serum from the midnight specimen is not being separated from the clot and refrigerated in a timely manner. b. Lactic acid will be present in serum that is not separated from the clot and will affect the freezing point osmolarity readings. c. If the laboratory is using a freezing point osmometer, results will be affected by alcohol ingestion; vapor pressure results would not be affected and could be used as a comparison. 5. a. Diabetes insipidus. b. Neurogenic diabetes insipidus. c. Nephrogenic diabetes insipidus. 1. 2. 3. 4. 5. 6. 7. 8. A D A D A A C C 9. 10. 11. 12. 13. 14. 15. 16. D A C B D A C B 17. 18. 19. 20. 21. 22. 23. 24. D D C B B A B D Case Studies and Clinical Situations 1. a. An elevated pH and a positive reagent strip reaction for nitrite. b. The reagent strip specific gravity would be much lower if the patient had been given radiographic dye. c. The reagent strip test for bilirubin would be positive. d. The reagent strip reaction for blood would be positive and red blood cells would be seen in the microscopic. 2. a. 1.018 b. Yes. c. It would agree with the reagent strip reading because like the osmometer, the reagent strip is not affected by high-molecular-weight substances. 3. Hemoglobin and myoglobin. a. Examine the patient’s plasma /serum. The breakdown of red blood cells to hemoglobin produces a red serum. Myoglobin is produced from skeletal muscle and is rapidly cleared from the plasma/serum. 4. a. Mrs. Smith has been eating fresh beets. b. Yes. The pH of Mrs. Smith’s urine is acidic or she has not recently consumed fresh beets. 5. No. The urine can contain increased pH, glucose, ketones, bilirubin, urobilinogen, nitrite, and small amounts of cellular structures. Chapter 5 Study Questions 1. 2. 3. 4. 5. 6. 7. A D A C D A D 8. 9. 10. 11. 12. 13. 14. B D 2,1,2,3,1,2,3 B A A D 15. 16. 17. 18. 19. 20. 21. B A C A A C A 3920_Ans_297-304 23/01/14 9:15 AM Page 299 Answers to Study Questions and Case Studies and Clinical Situations 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. B C A C B A D A C 1, 2, 1, 2, 1, 2 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. B A 1,3,4,2 A D C A C A B 42. 43. 44. 45. 46. 47. 48. 49. B D C B C C A C Case Studies and Clinical Situations 1. a. The blood glucose is elevated and has exceeded the renal tubular maximum (Tm) for glucose. b. Diabetes mellitus. c. It indicates diabetes mellitus related renal disease. d. Renal tubular reabsorption disorders. 2. a. Yellow foam. b. Possible biliary-duct obstruction preventing bilirubin from entering the intestine. c. Icteric. d. Protection from light. 3. a. Hemoglobinuria. b. Increased hemoglobin presented to the liver results in increased bilirubin entering the intestine for conversion to urobilinogen. c. The circulating bilirubin is unconjugated. d. It would if a multisix reagent strip is used and would not if a Chemstrip is used. A Watson-Schwartz test is more specific for porphobilinogen. 4. a. Negative chemical reactions for blood and nitrite. Ascorbic acid interference for both reactions. A random specimen or further reduction of nitrite could cause the negative nitrite. b. Glucose, bilirubin, LE. Ascorbic acid is a strong reducing agent that interfers with the oxidation reaction in the glucose test. Ascorbic acid combines with the diazo reagent in the bilirubin and LE tests, lowering the sensitivity. c. The dark yellow color may be caused by beta-carotene and vitamin A, and some B vitamins also produce yellow urine. d. Non-nitrite–reducing microorganisms; lack of dietary nitrate; antibiotic administration. 5. a. To check for possible exercise-induced abnormal results. b. Negative protein and blood, possible changes in color and specific gravity. c. Renal. 299 6. a. b. c. d. 7. a. No, the specimen is clear. Myoglobinuria. Muscle damage from the accident (rhabdomyolysis). Yes. Myoglobin is toxic to the renal tubules. Laboratory personnel are not tightly capping the reagent strip containers in a timely manner. b. Personnel performing the CLIA-waived reagent strip test are not waiting 2 minutes to read the LE reaction. c. The student is not mixing the specimen. d. The reagent strips have deterioated and the quality control on the strips was not performed prior to reporting the results. Chapter 6 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. A D C C A B C D C D D A C B C A D 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. D B C A B C D D B D A B C C D D B 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. A C A D A C D A A C D C 4, 3, 5, 1 3, 5, 2, 6, 4 4, 8, 7, 6, 1, 5 3, 5, 2, 1, 7, 4 Case Studies and Clinical Situations 1. a. Yeast grows best at a low pH with an increased concentration of glucose. b. Yes, this exceeds the renal threshold. c. No, yeast is not capable of reducing nitrate to nitrite. d. Moderate blood with no RBCs. e. Myoglobin is the cause of the positive chemical test result for blood. The patient has been bed-ridden for an extended period of time, causing muscle destruction. 3920_Ans_297-304 23/01/14 9:15 AM Page 300 300 Answers to Study Questions and Case Studies and Clinical Situations 2. a. The large objects are in a different plane from that of the urinary constituents. b. Contamination by artifacts. c. No, because they are in a different plane. d. Polarizing microscopy. 3. a. Renal tubules. b. Yes, viral infections can cause tubular damage. c. RTE cells absorb the bilirubin-containing urinary filtrate. d. Liver damage inhibits processing of reabsorbed urobilinogen. e. Hemolytic anemia. 4. a. The patient is taking a pigmented medication, such as phenazopyridine. b. Yes. c. Ask what medications the patient is taking. d. Ampicillin. 5. a. Calcium oxalate. b. Monohydrate and dihydrate calcium oxalate. c. Oval: monohydrate; envelope: dihydrate. d. Monohydrate. 6. a. Microscopic results do not match the chemical tests for blood, nitrite, and leukocyte esterase. b. The specimen has been unpreserved at room temperature for too long, the cells have disintegrated, and the bacteria have converted the nitrite to nitrogen. c. The pH. d. Ask the clinic personnel to instruct the patient to collect a midstream clean-catch specimen and have the specimen delivered immediately to the laboratory. 7. a. No, because they are associated with strenuous exercise. b. The positive blood reaction is from hemoglobinuria or myoglobinuria resulting from participating in a contact sport. The protein is orthostatic. c. Increased excretion of RTE cell lysosomes in the presence of dehydration. 8. a. Yes, the waxy casts are probably an artifact such as a diaper fiber. Waxy casts are not associated with negative urine protein. b. No, this is normal following an invasive procedure. c. Yes, tyrosine crystals are seen in severe liver disease; therefore, the bilirubin should be positive. The crystals may be an artifact or from a medication. d. Yes, uric acid crystals may be mistaken for cystine crystals. e. Yes, radiographic dye crystals associated with a high specific gravity resemble cholesterol crystals. f. No, Trichomonas is carried asymptomatically by men. g. No, calcium carbonate crystals are found in alkaline urine; therefore, clumps of amorphous phosphates may be present. Chapter 7 Study Questions 1. 2. 3. 4. 5. 6. 7. B C B C B D C 8. 9. 10. 11. 12. 13. 14. D A C C C B D 15. 16. 17. 18. 19. 20. A C A A A D Case Studies and Clinical Situations 1. a. Acute glomerulonephritis. b. M protein in the cell wall of the group A streptococcus. c. Glomerular bleeding. d. No, they are also passing through the damaged glomerulus. e. Good prognosis with appropriate management of secondary complications. f. Henoch-Schönlein purpura. 2. a. IgA nephropathy. b. Serum IgA level. c. Chronic glomerulonephritis/end-stage renal disease. d. Impaired renal tubular reabsorption associated with end-stage renal disease. e. The specific gravity is the same as that of the ultrafiltrate, indicating a lack of tubular concentration. f. The presence of extreme urinary stasis. 3. a. Nephrotic syndrome. b. Nephrotic syndrome may be caused by sudden, severe hypotension. c. Changes in the electrical charges of the shield of negativity produce increased membrane permeability. d. Decreased plasma albumin lowers the capillary oncotic pressure, causing fluid to enter the interstitial tissue. e. Reabsorption of filtered lipids by the RTE cells. 4. a. Minimal change disease. b. Nephrotic syndrome, focal segmental glomerulosclerosis. c. Good prognosis with complete remission. 3920_Ans_297-304 23/01/14 9:15 AM Page 301 Answers to Study Questions and Case Studies and Clinical Situations 5. a. Goodpasture syndrome. b. The autoantibody attaches to the glomerular capillaries, causing complement activation and destruction of the capillaries. c. Wegener’s granulomatosis. d. Antineutrophilic cytoplasmic antibody. e. Granuloma formation resulting from autoantibodies binding to neutrophils in the vascular walls and initiating an immune response. 6. a. Cystitis, UTI. b. The specimen is very dilute. c. Irritation of the urinary tract will cause a small amount of bleeding. The cells and bacteria may cause a trace protein or it may be a false-positive due to the high pH. d. Yes, glitter cells are seen in hypotonic urine. e. Female children. f. Pyelonephritis. 7. a. Intravenous pyelogram. b. Chronic pyelonephritis. c. WBC cast. d. Reflux nephropathy. e. Performing a Gram stain. f. Radiographic dye. g. Permanent tubular damage and progression to chronic, end-stage renal disease. 8. a. Abnormal. b. Acute interstitial nephritis. c. This disorder is an inflammation not an infection. d. Discontinue the medication because it is causing the allergic reaction. 9. a. Acute renal failure. b. The prerenal sudden decrease in blood flow to the kidneys. c. Lack of renal concentrating ability. d. Tubular damage. e. The increased diameter of the damaged distal convoluted tubule and extreme urinary stasis allowing casts to form in the collecting ducts. 10. a. Renal lithiasis. b. The high specific gravity. c. Yes, the dark yellow color and high specific gravity indicate a concentrated urine, which induces the formation of renal calculi. d. Calcium oxalate. e. Increased hydration and dietary changes. 301 Chapter 8 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. A C B C A A C B D 10. 11. 12. 13. 14. 15. 16. 17. 18. D D B C A D B, A, B, B, A D B 19. 20. 21. 22. 23. 24. 25. 26. 27. B B D D D C B D D, F, A, E, C, B Case Studies and Clinical Situations 1. a. b. c. d. 2. a. b. c. 3. a. b. c. d. e. 4. a. b. c. d. e. 5. a. b. c. 6. a. b. c. 7. a. b. c. d. e. Underdevelopment of the liver. Yes, with severe acquired liver disease. Tyrosine crystals; leucine crystals, bilirubin crystals. Protect the specimen from light. Isovaleric acidemia. Maple syrup urine disease. Yes, the MS/MS screen would be positive. Renal lithiasis. Impaired renal tubular reabsorption of cystine. Lysine, arginine, ornithine. They are more soluble than is cystine. The disorder is inherited. Yes. Yes, uric acid crystals accumulating on the surface of the diaper could have an orange color. Lesch-Nyhan disease. Yes, the disease is inherited as a sex-linked recessive. Hypoxanthine guanine phosphoribosyltransferase. Yes. The urine may contain melanin or homogentisic acid. Yes. Melanin will react with sodium nitroprusside, the reagent used on reagent strips for the detection of ketones. Yes. Homogentisic acid turns black in alkaline urine. Yes, the purple blue color could indicate the presence of indican in the urine. Hartnup disease. Good with proper dietary supplements. The Ehrlich reaction Acetylacetone. Porphobilinogen. Blood. Free erythocyte protoporphyrin (FEP). 3920_Ans_297-304 23/01/14 9:15 AM Page 302 302 Answers to Study Questions and Case Studies and Clinical Situations Chapter 9 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. B C B A B B, B, A, A C A C C D D 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. A B C D D D B D A B B B 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. C A C A C D B A A C C D Case Studies and Clinical Situations 1. a. Cerebral hemorrhage because of the presence of erythrophagocytosis, even distribution of blood, and patient’s history. b. No, they would be consistent with peripheral blood entering the CSF. c. No, they are consistent with the percentages seen in peripheral blood. d. Hemosidern granules and hemotoidin crystals. e. A traumatic tap. 2. a. An India ink preparation. b. Cryptococcus meningitis. c. Immunologic testing for Cryptococcus. d. Rheumatoid factor. e. Acid-fast staining and culture. f. Noticeable oligoclonal bands in both the CSF and serum. 3. a. CSF/serum albumin index = 6.7. b. Yes. c. IgG index = 1.5. d. Immunoglobulin synthesis within the CNS. e. Multiple sclerosis. f. Oligoclonal banding only in the CSF. g. Myelin basic protein. 4. a. Viral, tubercular, or fungal meningitis. b. No, the Gram stain would be negative in viral and tubercular and not always positive in fungal meningitis. c. Yes. Lymphocytes are very predominant in viral meningitis. d. Yes, a CSF lactate level of 25 mg/dL or less would aid in confirming bacterial meningitis. The lactate level would be higher in tubercular and fungal meningitis. 5. a. Stain precipitate is being confused with Grampositive cocci. b. Differentials are being reported from the counting chamber. c. The albumin is contaminated. d. The specimens are not being promptly delivered to the laboratory. Chapter 10 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. C D B C D A B D C B B 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. C A A B B A C A C A A 23. 24. 25. 26. 27. 28. 29. 30. 31. D B D B D B C A B Case Studies and Clinical Situations 1. a. b. c. d. 2. 3. 4. 5. 6. Sperm concentration, motility, and morphology. 21,000,000; no. 1,800,000; no. Yes. The normal sperm concentration is 20 to 60 million/mL. Spermatid counts over 1 million are considered abnormal. Both of these abnormal results and the abnormal motility are related to defects in sperm maturation. a. Male antisperm antibodies may form following vasovasostomy procedures. b. The MAR test and the immunobead test. c. The MAR test detects the presence of IgG male sperm antibodies. The immunobead test delineates the areas of the sperm (head, tail, neck) that are affected by the antibodies. d. Clumping, ovum penetration, and motility. The specimen contains urine, which is toxic to sperm, therefore decreasing viability. The specimen was improperly collected, and the first part of the ejaculation was lost. a. Yes, there is insufficient prostatic fluid present. b. Zinc, citrate, and acid phosphatase. c. Sperm motility is severely affected. a. Acid phosphatase and seminal glycoprotein p30 tests. b. Microscopic examination for the presence of sperm. 3920_Ans_297-304 23/01/14 9:16 AM Page 303 Answers to Study Questions and Case Studies and Clinical Situations Chapter 11 Case Studies and Clinical Situations Study Questions 1. 2. 3. 4. 5. B A A B B, C, B, A, D, B, D 6. A 7. B 8. B 9. 10. 11. 12. 13. 14. 15. 16. 17. 303 B C A A C A B B A 18. 19. 20. 21. 22. 23. 25. D B Negative C A C A Case Studies and Clinical Situations 1. a. 1. Sterile, heparinized tube, liquid EDTA tube, nonanticoagulated tube. b. MSU crystals are seen in gout. c. Highly birefringent, needle-shaped crystals under polarized light that turn yellow when aligned with the slow vibration of red compensated polarized light. d. Infection is frequently a complication of severe inflammation. 2. a. WBC diluting fluid containing acetic acid was used. b. Normal, hypotonic, or saponin-containing saline should be used. c. Crystal-induced inflammatory and septic. d. Gram stain and culture, crystal examination. 3. a. Noninflammatory. b. Hydroxyapatite crystals. c. Glucose. A normal result is consistent with noninflammatory arthritis. 4. a. Fibrinogen. b. EDTA or heparinized tube. c. No, the bacteria will be trapped in the clot. 1. a. Pleural fluid. b. Transudate, because all the test results are consistent with those of a transudate. c. Pleural fluid to serum ratios of cholesterol and bilirubin. 2. a. Pneumonia. b. Chest tube drainage. 3. a. 1.6. b. Transudate. The SAAG is above 1.1. c. Hepatic disorder. 4. a. To differentiate between cirrhosis and peritonitis; cirrhosis. b. Pancreatitis or gastrointestinal perforation; alkaline phosphatase. c. Rupture or accidental puncture of the bladder. d. To detect the presence of gastrointestinal (CEA) and ovarian (CA 125) cancers. 5. The patient has been a victim of blunt trauma and the physician wants to determine if abdominal bleeding is occurring; abdominal bleeding. 6. Thyroid profile; CA 125. Chapter 13 Study Questions 1. 2. 3. 4. 5. 6. 7. B C A C B A D 8. 9. 10. 11. 12. 13. 14. 2, 4, 1, 3 A C B True A True 15. 16. 17. 18. 19. 20. C True C B B D Case Studies and Clinical Situations Chapter 12 Study Questions 1. 2. 3. 4. 5. 6. 7. C D A D C D B, A, A, A, B, A, B 8. B 9. 10. 11. 12. 13. 14. 15. 16. 17. B C D D D C B A D 18. 19. 20. 21. 22. 23. 24. 25. 26. B C B B B C B D D 1. a. Yes. b. FLM. c. The level of phosphatidylglycerol present in the fetal lungs. d. Phosphatidylglycerol is essential for FLM, and levels do not always parallel lecithin levels in fetuses of diabetic mothers. 2. a. A neural tube disorder such as spina bifida or anencephaly. b. An acetylcholinesterase level. c. The amniotic fluid specimen contains blood. 3920_Ans_297-304 23/01/14 9:16 AM Page 304 304 Answers to Study Questions and Case Studies and Clinical Situations 3. a. b. c. d. 4. a. b. c. d. 5. a. False-positive result. False-positive result. No effect. False-positive result. False-positive result. False-positive result. False-positive or test interference. No effect. The specimen was exposed to light, possible wrong specimen sent. Chapter 14 3. a. Patient #1: gastric reflux medication containing bismuth may produce black stools; Patient #2: medications such as aspirin and other NSAIDs may cause gastric bleeding; Patient #3: red meat was not avoided for 3 days prior to sample collection. b. Provide dietary and medication instructions to patients. c. The Hemoccult ICT immunochemical test. 4. a. The APT test cannot be performed because the hemoglobin is already denatured. b. The pH will be low because increased carbohydrates are available for bacterial metabolism. c. The infant had ingested maternal blood. d. Yes, adequate carbohydrates are not present, and fats are being metabolized for energy. Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. C A C B D D D C D C 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. A C D B B False C C C D 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. B C B A B B A C C B Case Studies and Clinical Situations 1. a. Secretory diarrhea. b. Stool culture. c. Probable: Salmonella, Shigella, Campylobacter, Yersinia, E. coli; Improbable: Staphylococcus, Vibrio. d. Osmotic diarrhea. 2. a. Microscopic examination for fecal fats. b. Neutral fats stain directly and appear as large, orangered droplets; soaps and fatty acids appear as smaller orange-red droplets after pretreatment of the specimen with heat and acetic acid. c. Quantitative fecal fat test. d. Bulky and frothy. e. Muscle fiber screening and the gelatin test for trypsin. f. Muscle fiber: failure to include red meat in the diet; gelatin test: intestinal degradation of trypsin or the presence of trypsin inhibitors. g. Chymotrypsin or elastase I. Chapter 15 Study Questions 1. 2. 3. 4. 5. D B C A C 6. 7. 8. 9. 10. A B A C A 11. 12. 13. 14. 15. C B C A D Case Studies and Clinical Situations 1. a. Vaginal pH, saline and KOH wet preps, Gram stain. b. KOH will reveal budding yeast. c. Culture and DNA direct hybridization probe (Affirm VPIII). d. Vulvovaginal candidiasis caused by Candida albicans. e. Antifungal agents. 2. a. Trichomoniasis caused by Trichomonas vaginalis. b. Wet mount, vaginal pH, amine test from KOH prep, DNA probe (Affirm VPIII), OSOM Trichomonas Rapid Test. c. Metronidazole. d. Yes. e. Complications include low birth rate, premature rupture of membranes, preterm delivery during pregnancy. 3. a. Desquamative inflammatory vaginitis secondary to atrophic vaginitis. b. Reduced estrogen production in postmenopausal women. c. Hormone replacement therapy (estrogen). 3920_Abbreviations_305-306 23/01/14 9:14 AM Page 305 Abbreviations AABB A:C ACE AChE ADA ADH AFP AGN AHG AIDS AIN ALA ANA ANCA AOA ARF ART ASHI ATN BAL BAT BSI BUN BV CAP CASA CDC CEA CHP CLIA CLRW CLSI CNS COC COLA CPPD CPR CSF CTAB Cu2O CuSO4 CV DBDH DCT DI DIDNTB DIV American Association of Blood Banks albumin:creatinine [ratio] angiotensin-converting enzyme acetylcholinesterase adenosine deaminase antidiuretic hormone alpha-fetoprotein acute glomerulonephritis antihuman globulin acquired immunodeficiency syndrome acute interstitial nephritis ␣ -aminolevulinic acid antinuclear antibody antineutrophilic cytoplasmic antibody American Osteopathic Association acute renal failure assisted reproductive technology American Society of Histocompatibility and Immunogenetics acute tubular necrosis bronchoalveolar lavage bacterial antigen test body substance isolation blood urea nitrogen bacterial vaginosis College of American Pathologists computer-assisted semen analysis Centers for Disease Control and Prevention carcinoembryonic antigen chemical hygiene plan Clinical Laboratory Improvement Amendments clinical laboratory reagent water Clinical and Laboratory Standards Institute central nervous system chain of custody Commission on Laboratory Assessment calcium pyrophosphate dihydrate cardiopulmonary resuscitation cerebrospinal fluid cetyltrimethylammonium bromide cuprous oxide copper sulfate coefficient of variation diisopropyl benzene dihydroperoxide distal convoluted tubule diabetes insipidus (3⬘,3⬙, diodo 4⬘,4⬙-dihydroxy-5⬙,5⬙dinitrophenyl)-3,4,5,6-tetra-bromosulphonphtalein desquamative inflammatory vaginitis DNPH EDS EDTA eGFR ELISA EPS EQA EQC EU FAH FDA FEP fFN FISH FLM FOBT FSGS FTA-ABS GALT gFOBT GFR GI GTT 1-H NMR H+ H2PO4 Hb HBV HCV HCO3 HDN HICPAC 5-HIAA HIV HLA-B12 hpf HRCT HSV IBS IDMS IEF IEM IFE iFOBT IgA IgG IgM ISO 2,4-dinitrophenylhydrazine early dumping syndrome ethylenediaminetetraacetic acid estimated glomerular filtration rate enzyme-linked immunoabsorbent assay expressed prostatic secretions external quality assessment external quality control Ehrlich unit fumarylacetoacetate hydrolase Food and Drug Administration free erythrocyte protoporphyrin fetal fibronectin fluorescence in situ hybridization fetal lung maturity fecal occult blood testing focal segmental glomerulosclerosis fluorescent treponemal antibody-absorption galactose-1-phosphate uridyl transferase guaiac fecal occult blood test glomerular filtration rate gastrointestinal glucose tolerance test hydrogen nuclear magnetic resonance spectroscopy titratable acid/hydrogen ion hydrogen phosphate ion hemoglobin hepatitis B virus hepatitis C virus bicarbonate ion hemolytic disease of the newborn Healthcare Infection Control Practices Advisory Committee 5-hydroxyindoleacetic acid human immunodeficiency virus human leukocyte antigen-B12 high-power field high-resolution computerized tomography herpes simplex virus irritable bowel syndrome isotope dilution mass spectrophotometry isoelectric focusing inborn error of metabolism immunofixation electrophoresis immunochemical fecal occult blood test immunoglobulin A immunoglobulin G immunoglobulin M International Organization for Standardization 305 3920_Abbreviations_305-306 23/01/14 9:14 AM Page 306 306 Abbreviations IUI IVF JC KOH LAF LBC LD LE LED LIS lpf L/S MAR MBP MDRD MESNA MoM mOsm MPGN MSAFP MS/MS MSDS MSU MSUD NaCl NFPA NH4+ NIRS NKDEP NRBC NTD OD OSHA PAH PAMG-1 PB PCR PCT intra-uterine insemination in vitro fertilization Joint Commission potassium hydroxide lateral flow assay lamellar body count lactate dehydrogenase leukocyte esterase light-emitting diode laboratory information system low-power field lecithin-sphingomyelin ratio mixed agglutination reaction myelin basic protein modification of diet in renal disease mercaptoethane sulfonate sodium multiples of the median milliosmole membranoproliferative glomerulonephritis maternal serum alpha-fetoprotein tandem mass spectrophotometry Material Safety Data Sheet monosodium urate (uric acid) maple syrup urine disease sodium chloride National Fire Protection Association ammonium ion near-infrared spectroscopy National Kidney Disease Education Program nucleated red blood cell neural tube defect optical density Occupational Safety and Health Administration p-aminohippuric acid placental ␣ 1-microglobulin peripheral blood polymerase chain reaction proximal convoluted tubule PEP PG PKU PM POC PPE PPMT PSA PSP PT QA QC RA RAAS RBC RCF RDS RF RGE RPM RPR RTE SAAG SD SKY SLE SSA STI TAT Tm TmG TMB UP UTI VDRL WBC WHO postexposure prophylaxis phosphatidyl glycerol phenylketonuria preventive maintenance point of care personal protective equipment pre- and post-massage test prostate-specific antigen phenolsulfonphthalein proficiency testing quality assurance quality control rheumatoid arthritis renin-angiotensin-aldosterone system red blood cell relative centrifugal force respiratory distress syndrome rheumatoid factor rapid gastric emptying revolutions per minute rapid plasma reagin renal tubular epithelial (cells) serum-ascites albumin gradient standard deviation fluorescent mapping spectral karyotyping systemic lupus erythematosus sulfosalicylic acid sexually transmitted infection turnaround time maximal reabsorptive capacity/tubular reabsorptive maximum maximal tubular reabsorption capacity for glucose 3,3⬘,5,5⬘-tetramethylbenzidine Universal Precautions urinary tract infection Venereal Disease Research Laboratory white blood cell World Health Organization 3920_Glossary_307-314 23/01/14 10:48 AM Page 307 Glossary 2-hour postprandial specimen Fractional collection specimen; urine specimen collected 2 hours after eating accreditation The process by which a program or institution documents meeting established guidelines accuracy Closeness of the measured result to the true value acholic stools Pale-colored stools acrosomal cap Tip of a spermatozoa head, which contains enzymes for entry into an ovum active transport Movement of a substance across cell membranes into the bloodstream by electrochemical energy acute interstitial nephritis Inflammation of the renal interstitium and tubules frequently caused by a reaction to a medication acute phase reactants Low-molecular-weight plasma proteins associated with infection and inflammation acute tubular necrosis Disorder affecting the renal tubular cells caused by decreased renal blood flow or toxic substances adjusted body weight Actual body weight compared with ideal body weight to correct for weight attributed to fat and not muscle aerosol Fine suspension of particles in air afferent arteriole A small branch of the renal artery through which blood flows to the glomerulus of the kidney albinism An inherited condition marked by decreased production of melanin albuminuria Protein (albumin) in the urine aldosterone A hormone that regulates reabsorption of sodium in the distal convoluted tubule alimentary tract The digestive tract, including structures between the mouth and the anus alkaptonuria Homogentisic acid in the urine caused by a failure to inherit the gene responsible for the production of homogentisic acid oxidase aminoacidurias Disorders in which increased amino acids are present in the urine amniocentesis Transabdominal puncture of the uterus and amnion to obtain amniotic fluid amnion The membranous sac that contains the fetus and amniotic fluid amniotic fluid Liquid that surrounds the fetus during gestation amyloid material A starch-like protein-carbohydrate complex that is deposited abnormally in tissue in some chronic disease states anaphylaxis Severe reaction caused by an autoimmune reaction to certain antigenic compounds andrology The study of diseases of the male reproductive organs antidiuretic hormone Hormone produced by the hypothalamus to regulate water reabsorption in the collecting duct antiglomerular basement membrane antibody Autoantibody against alveolar and glomerular capillary basement membranes found in Goodpasture syndrome antineutrophilic cytoplasmic antibody Autoantibody that reacts with neutrophils in the renal tubules. Provides a laboratory test for Wegener granulomatosis. anuria Complete stoppage of urine flow arachnoid granulations Projections on the arachnoid membrane of the brain through which cerebrospinal fluid is reabsorbed arthritis Inflammation of the synovial joints arthrocentesis The puncture of a joint to obtain synovial fluid ascites Abnormal accumulation of peritoneal fluid ascitic fluid Watery fluid that accumulates in the peritoneal cavity in certain disease conditions astrocytomas Tumors of the brain and spinal cord atrophic vaginitis Syndrome in postmenopausal women caused by reduced estrogen production azotemia Increased nitrogenous waste products in the blood bacterial endocarditis Inflammation of the endocardial membrane of the heart caused by bacterial infection bacterial vaginosis Inflammation of the vagina most often caused by Gardnerella vaginalis bacteriuria Bacteria in the urine beta2 microglobulin A subunit of the class I major compatibility antigens that enters the blood at a constant rate basal cells Cells located in the basal layer of the vaginal stratified epithelium bilirubin A bright yellow pigment produced in the degradation of heme biliverdin Green pigment formed by the oxidation of bilirubin biohazardous Pertaining to a hazard caused by infectious organisms birefringent The ability to refract light in two directions blood–brain barrier The barrier between the brain tissue and capillary blood that controls the passage of substances in the blood to the brain and cerebrospinal fluid body substance isolation A guideline stating that all moist body substances are capable of transmitting disease Bowman’s capsule Part of the nephron that contains the glomerulus bright-field microscopy A procedure by which magnified images appear dark against a bright background bulbourethral glands Two small glands located on each side of the prostate gland 307 3920_Glossary_307-314 23/01/14 10:48 AM Page 308 308 Glossary carcinogenic Capable of causing cancer casts Elements excreted in the urine in the shape of renal tubules catheterized specimen A urine specimen collected by passing a sterile tube into the bladder chain of custody Step-by-step documentation of the handling and testing of legal specimens chain of infection A continuous link in the transmission of harmful microorganisms between a source and a susceptible host chemical hygiene plan Protocol established for the identification, handling, storage, and disposal of all hazardous chemicals chemical sieving Macroscopic screening of urine to determine the need for a microscopic examination choroid plexuses A network of capillaries in the ventricle of the brain that produces cerebrospinal fluid chylous material A milky lymphatic fluid that contains triglycerides and chylomicrons cirrhosis Chronic liver disease that results in loss of liver cell function clarity Transparency of urine, ranging from clear to turbid Clinical Laboratory Improvement Amendments Governmental regulatory agency that establishes quality standards for laboratory testing Clinical and Laboratory Standards Institute (CLSI) Nonprofit organization that publishes recommendations for laboratory tests clue cells Squamous epithelial cells covered with the gramnegative bacteria Gardnerella vaginalis coefficient of variation Standard deviation expressed as a percentage of the mean collecting duct Part of the nephron where the final concentration of urine takes place through the reabsorption of water colligative property Freezing point, boiling point, vapor pressure, or osmotic pressure property that is mathematically proportional to the concentration of a solution compensated polarized light Polarized light, using a compensator to separate light rays into slow- and fast-moving vibrations constipation Infrequent production of feces that results in small, hard stools continuous quality improvement An institutional program that focuses on customer satisfaction and expectations control mean Average of all data points control range Limit within which expected control values lie, usually plus or minus two standard deviations from the mean countercurrent mechanism A selective urine concentration process in the ascending and descending loops of Henle creatinine A substance formed by the breakdown of creatine during muscle metabolism creatinine clearance A test used to measure the glomerular filtration rate crenated Shrunken and irregularly shaped or notched cylindruria The presence of urinary casts cystatin C Small protein produced at a constant rate by all nucleated cells cystinosis An inherited recessive disorder that disrupts the metabolism of cystine cystinuria Cystine in the urine that occurs as a result of a defect in the renal tubular reabsorption of amino acids cystitis An inflammation of the bladder cytogenetic analysis An analysis of cellular chromosomes dark-field microscopy Microscopic technique by which magnified images appear bright against a dark background D-dimer A product of fibrinolysis demyelination The destruction of the myelin sheath that protects a nerve density Concentration of solutes present per volume of solution desquamative inflammatory vaginitis Syndrome characterized by purulent vaginal discharge, vaginal erythema, and dyspareunia diarrhea Watery stools diarthroses Freely movable joints disinfectant A substance that destroys microorganisms that is used on surfaces rather than the skin distal convoluted tubule Part of the nephron between the ascending loop of Henle and the collecting duct where the final concentration of urinary filtrate begins dysentery An inflammation of the intestines that is caused by microorganisms and results in diarrhea dysmorphic Irregularly shaped dyspareunia Abnormal pain during sexual intercourse dyspnea Difficulty breathing dysuria Painful urination edema An accumulation of fluid in the tissues efferent arteriole The small renal artery branch through which blood flows away from the glomerulus effusion An accumulation of fluid between the serous membranes electronic quality control Mechanical or electrical sample used in place of a liquid control to verify reliability of test results endogenous procedure A test that uses a substance originating within the body epididymis Small structure that forms the first part of the secretory duct of the testes erythrophagocytosis Engulfment of red blood cells by macrophages examination variable Processes that occur during testing of a sample exogenous procedure A test that requires a substance to be infused into the body external quality assessment Use of quality control materials from an outside agency to monitor reliability of test results external quality control Commercial controls used to verify accuracy and reliability of patient test results exudate Serous fluid effusion caused by conditions producing damage to the serous membranes 3920_Glossary_307-314 23/01/14 10:48 AM Page 309 Glossary Fanconi syndrome A group of disorders marked by renal tubular dysfunction associated with some inherited and acquired conditions fasting specimen The second voided urine specimen collected after fasting ferritin A major storage form of iron found in the liver, spleen, and bone marrow fetal lung maturity The presence of a sufficient amount of surfactant lipoproteins to maintain alveolar stability first morning specimen The first voided urine specimen collected immediately upon arising; recommended screening specimen flatus Gas expelled from the anus fluorescent microscopy Microscopic technique by which naturally fluorescent substances or those that have been stained by a fluorescent dye produce an image when illuminated with a light of a specific wavelength focal segmental glomerulosclerosis Disorder that affects only certain areas of the glomerulus and produces heavy proteinuria fomite Inanimate object that can serve as a reservoir for pathogenic organisms free water clearance A test to determine the ability of the kidney to respond to the state of body hydration Froin syndrome Failure of spinal fluid in the spinal canal to mix with the cerebrospinal fluid in the ventricles, producing xanthochromic, rapidly clotting cerebrospinal fluid fructosuria The presence of fructose in the urine galactosuria The presence of galactose in the urine Gardnerella vaginalis Rod-shaped bacteria that causes bacterial vaginitis gastrocolic fistula Abnormal passageway between the stomach and the colon ghost cells Red blood cells that have lost their hemoglobin, leaving only the cell membrane; appear in hyposthenuric urine glans The gland-like body at the tip of the penis glomerular filtration barrier Structure of the walls of the glomerular capillaries that prevents the filtration of large molecules from the blood into the urine filtrate glomerular filtration rate The volume of plasma that is filtered by the glomerulus in a specified time glomerulonephritis An inflammation of the glomerulus that results in impaired glomerular filtration glomerulosclerosis The destruction of glomeruli by scarring and fibrin deposition glomerulus Tuft of capillary blood vessels located in Bowman’s capsule where filtration occurs gout Disorder related to elevated serum uric acid that results in the accumulation of uric acid crystals in a moveable joint glucose tolerance specimens Fractional collection specimens; urine specimens are collected at the same time blood samples are drawn to compare the levels of glucose in blood and urine glycogenesis The conversion of glucose to glycogen 309 glycogenolysis The conversion of glycogen to glucose glycosuria Glucose in the urine (glucosuria) granuloma Modular accumulation of inflammatory cells Guillain-Barré syndrome Autoimmune disorder that causes destruction of the myelin sheath that surrounds the peripheral nerves, resulting in loss of motor function harmonic oscillation densitometry A method of measuring specific gravity by measuring the change in the frequency of a sound wave after it enters a solution Hartnup disease A recessive inherited disorder marked by intestinal absorption abnormalities and renal aminoaciduria hematoidin Yellow, crystalline substance that results from the destruction of red blood cells hematuria Blood in the urine hemoglobinuria Hemoglobin in the urine hemolytic disease of the newborn Rh incompatibility between mother and fetus that can cause hemolysis of the fetal red blood cells hemoptysis Blood in the sputum hemosiderin An insoluble form of storage iron; a product of red blood cell hemolysis hemothorax The accumulation of blood in the pleural cavity homocystinuria The presence of homocystine in the urine caused by an inherited autosomal recessive disorder hyaluronic acid Glycosaminoglycan found in synovial fluid that provides lubrication to the joints hydramnios Excess amniotic fluid hydrostatic pressure Pressure exerted by a liquid hyperglycemia Elevated glucose levels in the blood hypernatremia Elevated blood sodium levels hypersthenuric Pertaining to urine specific gravity greater than the 1.010 of the glomerular filtrate hyponatremia Decreased blood sodium levels hyposthenuric Pertaining to urine specific gravity lower than the 1.010 of the glomerular filtrate hypoxia Lack of oxygen iatrogenic Pertaining to a condition caused by treatment, medications, or diagnostic procedures ideal body weight Statistical calculation of body weight related to height IgA nephropathy Damage to the glomerular membrane caused the deposition of IgA immune complexes on the membrane immune complexes Antigen-antibody combinations inborn error of metabolism Failure to inherit the gene to produce a particular enzyme indicanuria The presence of indican in the urine infection control Procedures to control and monitor infections within an institution infertility The inability to conceive interference-contrast microscopy A procedure by which three-dimensional images of a specimen are obtained internal quality control Electronic, internal, and procedural controls contained within the test system that ensures the reliability of the test system interstitial Pertaining to spaces between tissue cells 3920_Glossary_307-314 23/01/14 10:48 AM Page 310 310 Glossary inulin A fructose-derived substance that is filtered by the kidney and not reabsorbed or secreted and that can be used to measure the glomerular filtration rate in vitro fertilization Fertilization between an ovum and a sperm performed in the laboratory ischemia Deficiency of blood to a body area isosthenuric Pertaining to urine specific gravity the same as the 1.010 of the glomerular filtrate jaundice Yellow appearance of skin, mucous membranes, and eye sclera due to increased amounts of bilirubin in the blood juxtaglomerular apparatus Specialized cells located on the afferent arteriole that regulate secretion of renin ketonuria Ketones in the urine Kohler illumination Adjustments made to the microscope condenser when objectives are changed labia The outer folds of the vagina lactobacilli A group of gram-positive rod-shaped bacteria lactosuria The presence of lactose in the urine lamellar bodies Organelles produced by type II pneumonocytes in the fetal lung that contain lung surfactants Langerhans cells Pancreatic cells lecithin Phospholipid that forms part of the cell wall used to determine fetal lung maturity lecithin-sphingomyelin ratio A comparison of lung surfactants that is performed to determine fetal lung maturity Lesch-Nyhan disease An inherited sex-linked recessive purine metabolism disorder marked by excess uric acid crystals in the urine leukocyturia Leukocytes (white blood cells) in the urine lipophages Macrophages that have ingested fat globules seen in peritoneal fluid liquefaction The conversion of solid or coagulated material to a liquid form lithiasis The formation of renal calculi (kidney stones) lithotripsy A procedure that uses ultrasonic waves to crush renal calculi loops of Henle The U-shaped part of the renal tubule that consists of a thin descending limb and a thick ascending limb lysosomes Cellular organelles that contain digestive enzymes macula densa Specialized cells located on the distal convoluted tubule that interact with the juxtaglomerular cells malabsorption Impaired absorption of nutrients by the intestine maldigestion Impaired digestion of foodstuffs maple syrup urine disease An autosomal recessive trait that causes increased levels of the branched-chain amino acids, leucine, isoleucine, valine, and their ketone acids in the urine Material Safety Data Sheet A document provided by the vendor or manufacturer of a chemical substance that describes the chemical’s characteristics maximal reabsorptive capacity The maximum reabsorption ability for a solute by renal tubules meconium The dark-green mucus-containing stool formed by a fetus medullary interstitium Spaces between the cells in the medulla of the kidney that contain highly concentrated fluid medulloblastomas Malignant tumor of the fourth ventricle and cerebellum melanoma A tumor of the melanogen-producing cells, which is frequently malignant melanuria Increased melanin in the urine melituria Increased urinary sugar meninges Protective membranes around the brain and spinal cord meningitis Inflammation of the meninges, frequently caused by microbial infection mesothelial cells Cells that line the serous membranes metabolic acidosis A decrease in the blood pH caused by a metabolic increase in acidic elements microalbuminuria Low levels of urine protein that are not detected by routine reagent strips midstream clean-catch specimen Specimen collected in a sterile container after cleansing the glans penis or urinary meatus; the first portion of urine is voided into the toilet, the midportion is collected, and the remaining portion is voided into the toilet Mobiluncus spp. Curved gram-variable rod-shaped bacteria mucin Glycoprotein found in mucus and in the skin, connective tissues, tendons, and cartilage mucopolysaccharides Glycosaminoglycans that consist of a protein core with polysaccharide branches mucopolysaccharidoses A group of genetic disorders marked by excess mucopolysaccharides in blood and urine multiple myeloma Malignant disorder that results in infiltration of the bone marrow by plasma cells myoglobin Iron-containing protein found in muscle tissue myoglobinuria Myoglobin in the urine necrosis Death of cells nephron A functional unit of the kidney that forms urine nephropathy Disease of the kidneys nephrotic syndrome A renal disorder marked by massive proteinuria, lipiduria, and edema caused by disruption of the glomerular membrane neutrophages Vacuolated macrophages containing phagocytized neutrophils; formerly called Reiter cells nocturia Excessive urination during the night occult blood Blood that is not visible to the naked eye Occupational Safety and Health Administration The government agency created to protect employees from potential health hazards in the workplace through the development and monitoring of regulations oligoclonal bands Electrophoretic bands migrating in the gamma region that are present in cerebrospinal fluid and serum oligohydramnios Decreased amniotic fluid oliguria A marked decrease in urine flow oncotic pressure The osmotic pressure of a substance in solution caused by the presence of colloids organic acidemias The accumulation of organic acids in the blood, mainly isovaleric, propionic, and methylmalonic acids 3920_Glossary_307-314 23/01/14 10:48 AM Page 311 Glossary orthostatic proteinuria Increased protein in urine only when an individual is in an upright position osmolar clearance The amount of plasma filtered each minute to produce a urine with the same osmolarity as plasma osmolality The osmotic pressure of a solution expressed in milliosmoles per liter; it is only affected by the number of particles present osmolarity The osmotic pressure of a solution expressed in milliosmoles per kilogram; it is affected only by the number of particles present osmotic diarrhea An increased retention of water and solutes in the large intestine associated with malabsorption and maldigestion osmotic gradient The difference in the concentration of substances on either side of a membrane outcomes Results of the process to improve customer satisfaction pancreatic insufficiency The decreased ability of the pancreas to secrete digestive enzymes parabasal cell Cells located in the luminal squamous epithelium of the vaginal mucosa paracentesis Surgical puncture into the abdominal cavity to obtain peritoneal fluid parietal membrane Serous membrane that lines the walls of the pleural, pericardial, and peritoneal cavities passive transport Movement of molecules across a membrane by diffusion because of a physical gradient pentosuria The presence of pentose sugars in the urine pericardiocentesis Surgical puncture into the pericardial cavity to obtain pericardial fluid pericarditis An inflammation of the membranes enclosing the heart peritoneal lavage Introduction and subsequent removal of fluid into the peritoneal cavity to
